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Forensic DNA Fingerprinting Kit Instruction Manual
Catalog #166-0007EDU explorer.bio-rad.com
The kit is shipped at room temperature. Open immediately upon arrival and store reagent bag at –20°C within 3 weeks of receipt.
Duplication of any part of this document is permitted for classroom use only. Please visit explorer.bio-rad.com to access our selection of language translations for Biotechnology Explorer kit curriculum.
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Can DNA evidence solve human problems? DNA fingerprinting is now used routinely to solve crimes. In recent years, news stories have reported how miniscule amounts of DNA have been used to identify individuals involved in incidents even many years in the past, as well as exonerate innocent people from incrimination. The power of DNA as a tool for individual identification captures students’ imaginations. This activity provides in-depth instruction about how restriction enzymes cleave DNA, how electrophoresis is used to separate and visualize DNA fragments, and how these techniques can be combined to obtain a DNA fingerprint. Principles of restriction analysis, plasmid mapping and DNA fragment size determination can also be documented with this kit. Open the door to rich discussions about scientific, ethical, and legal implications of DNA profiling. DNA fingerprinting is used in medical and forensic procedures, as well as in paternity determinations to discern genetic relationships between individuals at the molecular level. This kit allows students to play the role of a forensic scientist and make a positive ID—that is, to simulate using real DNA as evidence and figure out for themselves: “Who done it?” In this activity, students analyze six different samples of plasmid DNA. One sample collected from a hypothetical “crime scene” and five samples obtained from “suspects” are digested with two restriction enzymes. The resulting DNA fragments are separated and visualized in agarose gels using Bio-Rad’s Fast Blast™ DNA staining solution. Based on the restriction fragment patterns, students compare the evidence and match one of the suspects’ DNA to the sample collected at the crime scene. As an alternative to the classical human forensic applications for this kit, have your students imagine they are high tech pathologists investigating an outbreak of an aggressive infectious disease that has never been seen before. The Centers for Disease Control and Prevention suspects that a new strain of bacteria has arisen that not only is the cause of the new disease, but also has acquired multiple resistance plasmids from some other bacterial strains. Their job is to develop a DNA diagnostic tool for identifying the culprit plasmids. They decide to use restriction enzyme analysis and “DNA electrophoresis fingerprinting” to identify and distinguish different suspect plasmids and track their spread through the environment. DNA from the cultures of a number of stricken patients has been isolated. Have your students identify the new killer bug before the pathogen gets out into the general population and starts a true epidemic! We strive to continually improve our Biotechnology Explorer kits and curricula. Please share your stories, comments and suggestions! You can download this complete instruction manual on the Internet. Visit us on the Web at explorer.bio-rad.com or call us in the US at 1-800-4BIORAD (1-800-424-6723). This curriculum was developed in collaboration with Len Poli and Russ Janigian of the S.F. Base Biotechnology Program in San Francisco, California, and Peggy Skinner of the Bush School in Seattle, Washington. We’d like to thank them for their invaluable guidance and contributions to this curriculum. Ron Mardigian Biotechnology Explorer Program Bio-Rad Life Science Group 2000 Alfred Nobel Drive Hercules, California 94547
Create context. Reinforce learning. Stay current. New scientific discoveries and technologies create more content for you to teach, but not more time. Biotechnology Explorer kits help you teach more effectively by integrating multiple core content subjects into a single lab. Connect concepts with techniques and put them into context with real-world scenarios.
• Use of restriction enzymes and electrophoresis to fingerprint DNA • Use of experimental controls • Interpretation of experimental results • Use of DNA evidence in court • Creation and use of standard curves
Environmental and Health Science
• Epidemiology and disease • Genetic testing • Role, place, limits, and possibilities of science and technology • Privacy of information issues
Cell and Molecular Biology
Chemistry of Life
• DNA structure, function, and chemistry • Chemistry of DNA electrophoresis • Biochemistry of restriction enzymes
• Cell structure and organization • Tissue types for biological sampling • Plasmid mapping
• Genetic variations in the human genome • Genotype vs. phenotype • Biodiversity • Bacterial defense mechanisms
• Restriction fragment length polymorphisms (RFLP analysis) • Genetic engineering techniques • Mendelian inheritance • Plasmid mapping
Table of Contents
Kit Inventory Checklist Kit Components and Required Accessories ..............2
Instructor’s Manual Background
Setting the Stage for Your Students ..........................4
Instructor’s Advance Preparation Guide ..............................................9
Implementation Timeline Workstation Checklist Instructor’s Advance Preparation Advance Preparation and Student Lessons ..............9 Student and Instructor Lab Setups ..........................11 Lab Prep and Lesson Highlights..............................13 Graphic Laboratory Protocol ..................................20
Quick Guide Student Manual
Pre-Lab Activity Lesson 1 Lesson 2 Post-Lab Activity Extension Activity 1 Extension Activity 2
Introduction to DNA Fingerprinting ..........................23 Restriction Digestion of DNA Samples ....................25 Agarose Gel Electrophoresis of DNA Samples........31 Analysis and Interpretation of Results ....................40 Plasmid Mapping ....................................................47 Constructing a Plasmid ..........................................55
Appendix A Appendix B Alternative DNA Fingerprinting Scenarios ..............60 Prelab Activities ......................................................63 A Review of Restriction Enzymes......................63 A Review of Electrophoresis..............................68 Instructor’s Answer Guide ......................................70 Fast Gel Protocol ....................................................90 Plasmid Maps ........................................................91 References ............................................................95
Appendix C Appendix D Appendix E Appendix F
6) Use critical thinking to solve problems.Overview for the Instructor Intended Audience This investigation is intended for use by any high school or college student. Students should wash their hands with soap before and after this exercise. If any of the solutions gets into a student's eyes. Storage Temperatures The kit is shipped at room temperature. 4) Have a clear understanding of the thought processes involved in scientific work. Safety Issues Eating. We have found that this approach allows a larger number of the diverse population of students we work with to experience the goals that have been identified above. The actual scenario employed is up to the discretion of the instructor. if so desired. drinking. Teaching Strategies This curriculum is designed to simulate human forensic testing. independent of the degree of prior familiarity with the chemistry of nucleic acids. smoking. The analysis sections of this investigation are intended to guide students through the process of discovering and understanding concepts that are of significance to the procedures and the analysis of the data at each step along the way. 1 . Open immediately upon arrival and store the reagent bag at –20°C within 3 weeks of receipt. flush with water for 15 minutes. 3) Weigh evidence and be able to analyze and interpret the data that are generated in this investigation with clarity and confidence. some degree of self pacing is possible. Student Objectives That all students who participate in this investigation: 1) Become challenged by the task and intrigued by the methodology of the investigation. but can also be used to simulate a wide range of applications for genetic analysis. 2) Develop an understanding of some of the basic scientific principles involved in DNA fingerprinting and analysis of plasmid mapping. and applying cosmetics are not permitted in the work area. Lab coats or other protective clothing should be worn to avoid staining clothes. Refer to alternative scenarios in Appendix A. 5) Develop the curiosity and confidence to further explore questions and issues involving scientific investigations. Wearing protective eyewear and gloves is strongly recommended. latex or vinyl gloves should be worn while handling the stain to keep hands from becoming stained. So long as the teacher has the opportunity to check on the progress and level of understanding of each group. Although Fast Blast DNA stain is not toxic. It is hoped that this approach (as compared to the teacher giving the students all of the background information) will make the entire investigation more comprehensible to a greater number of students.
100–1000 µl. 100 µl 1 vial 10. Suspect 3 (S3) DNA with buffer. Gel staining trays 4 Required Accessories Number/Kit Adjustable micropipet.000 units 8. Fast Blast DNA stain. catalog #166-4000EDU 1–8 Power supply. catalog #166-0507EDU 1 Adjustable micropipet. EcoRI/PstI. 5 g 1 15. catalog #166-0501EDU Gel support film (50 sheets). Suspect 5 (S5) DNA with buffer. Kit Components Number/Kit 1.5 ml 30 14. lyophilized. 100 ml 1 16. Suspect 2 (S2) DNA with buffer. catalog #166-0508EDU 1 Pipet tips.5 ml 1 vial 9. restriction enzyme mix. Colored micro test tubes. 5 racks of 200. catalog #166-0602EDU or mini centrifuge Rocking platform. 500x. lyophilized. 2–20 µl. We recommend that students be teamed up – two to four students per workstation. 100 ml 1 vial 12. Electrophoresis buffer. 2. 3. Agarose powder. Sterile water. Suspect 1 (S1) DNA with buffer. catalog #223-9347EDU 1 Horizontal electrophoresis chamber. catalog #166-0504EDU or Mini incubation oven. lyophilized. 1. catalog #166-0506EDU 1–8 Pipet tips. 60 µg 1 vial 4. HindIII lambda digest (DNA size marker). 100–1000 µl. catalog #164-5050EDU 1–4 Adjustable micropipet. catalog #170-2984EDU Microcentrifuge. lyophilized. Foam micro test tube holders 16 17. 60 µg 1 vial 1 vial 7. 60 µg 1 vial 2. 20–200 µl.Kit Inventory Checklist This section lists the equipment and reagents necessary to conduct the Forensic DNA Fingerprinting laboratory. lyophilized. lyophilized. Suspect 4 (S4) DNA with buffer. 60 µg 1 vial 5. 2. Clear micro test tubes. catalog #166-0709EDU Number/Kit 1 1 1 1 ✔ (✔) ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ ✔ (✔) ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ ✔ (✔) ❑ ❑ ❑ ❑ 2 . catalog #223-9350EDU 1 Permanent markers 1 Microwave oven or hot plate 1 Distilled water 1 500 ml Erlenmeyer flask for microwaving agarose 1 500 ml flask or beaker for diluting DNA stain 1 Ice bucket with ice 1 Laboratory tape (not Scotch 3M brand or similar tape) 1 Optional Accessories 37°C water bath.2 µg/µl. 2–200 µl 5 racks of 200. 60 µg 1 vial 6. 60 µg 1 vial 3. Crime Scene (CS) DNA with buffer.0 ml 60 13. DNA sample loading dye 1 vial 11. 0. 50x TAE. lyophilized.
100 ml. 500x. run & stain 48 1% or 16 3% 7x10 cm agarose gels) Medium DNA Electrophoresis Reagent Pack. catalog #166-0420EDU Molecular biology agarose. DNA markers. catalog #166-0047EDU Small DNA Electrophoresis Reagent Pack. EcoRI/PstI restriction enzyme mix. run & stain 1080 1% or 360 3% 7x10 cm agarose gels) 3 . catalog #166-0027EDU (contains crime scene and suspect DNA samples. run & stain 270 1% or 90 3% 7x10 cm agarose gels) Large DNA Electrophoresis Reagent Pack. catalog #166-0455EDU (to pour. sterile water) EcoRI/PstI restriction enzyme mix. catalog #166-0460EDU (to pour. catalog #161-3100EDU DNA fingerprinting kit refill package. Fast Blast DNA stain. sample loading buffer. 25 g. catalog #166-0450EDU (to pour.Refills Available Separately Fast Blast DNA stain.
First described by English geneticist Alec Jeffries in 1985. Restriction enzymes were named before scientists understood how they functioned because they would limit (or restrict) the growth of phages. The length of each fragment will depend upon the location of restriction sites on the DNA molecule. such as the samples included in this kit. RFLP analysis provides a unique banding pattern based on the restriction sites present in an individual’s DNA sequence. fragments of varying sizes are produced. It may be important for you to point out to your students that this laboratory exercise models the more elaborate technique that is performed on complex human DNA samples. When restriction enzymes are used to cut strands of circular plasmid DNA. Restriction enzymes can be used to cut DNA isolated from any source. a restriction enzyme will make a cut at each of those sites. resulting in multiple fragments. If a specific restriction site occurs in more than one location on a DNA molecule. A restriction enzyme acts like molecular scissors. These restriction endonucleases (endo = within. EcoRI was isolated from Escherichia coli.INSTRUCTOR'S MANUAL BACKGROUND Instructor’s Manual Background Introduction Technicians working in forensic labs are often asked to do DNA profiling or “fingerprinting” to analyze evidence in law enforcement cases and other applications. Bacterial restriction enzymes recognize very specific DNA sequences within the phage DNA and then cut the DNA at that site. A restriction enzyme sits on a DNA molecule and slides along the helix until it recognizes specific sequences of base pairs that signal the enzyme to stop sliding. The patterns in this exercise are produced from one sample that represents DNA taken at the crime scene and five samples obtained from suspects in the case. nuclease = enzyme that cuts nucleic acids) are named for the bacteria from which they were isolated. if a linear piece of DNA is cut with a restriction enzyme whose specific recognition site is found at two different locations on the DNA molecule. In this lab activity. Restriction Enzymes Scientists have benefited from a natural. Although the bacteria’s own DNA may also contain these sites the bacteria protect their own restriction sites by adding a methyl group. Phages are viruses that infect and destroy bacteria. the result will be three fragments of different lengths. For example. DNA profiling involves polymerase chain reaction (PCR) amplification which allows the analysis of minute quantities of DNA in a much shorter time. Currently. students will compare band patterns produced by restriction enzyme cleavage of DNA samples when separated on an agarose gel (RFLP). Instructor’s Manual 4 . Once purified in the laboratory the fragmented phage DNA no longer poses a threat to the bacteria. Restriction Fragment Length Polymorphism (RFLP) has been the workhorse of forensic DNA profiling for many years. the result will be two fragments of different lengths. Therefore. The term electrophoresis means to carry with electricity. bacterial defense mechanism: the restriction enzyme. These enzymes destroy DNA from invading viruses. making cuts at specific sequence of base pairs that it recognizes. or bacteriophages (phages). DNA that has been cut with restriction enzymes can be separated and observed using a process known as agarose gel electrophoresis. The resulting fragments can then be used to create a plasmid map – see page 47. The enzyme then cuts or chemically separates the DNA molecule at that site—called a restriction site. If the piece of DNA is circular and is cut with a restriction enzyme whose specific recognition site is found at two different locations on the DNA molecule.
identify. the relative positions of radiolabeled bands in a gel are determined by the size of the DNA fragments in each band. This probe can be described as a "radioactive tag" that will bind to a single stranded DNA fragment and produce a band in a gel or a band on a piece of blotting membrane that is a replica of the gel (also known as a Southern blot). body fluids (blood and semen). DNA Fingerprinting Each person has similarities and differences in DNA sequences. The matrix of the agarose gel acts as a molecular sieve through which smaller DNA fragments can move more easily than larger ones. The DNA is then treated with restriction enzymes that cut the DNA into fragments of various length. Imagine that all the desks and chairs in the classroom have been randomly pushed together. Because of its specificity. A direct current is passed between wire electrodes at each end of the chamber. Consider this analogy. such as blood stains or mummified tissue. they will be drawn toward the positive pole (anode) when placed in an electric field. DNA analysis can even be done from dried material. DNA fragments are loaded into an agarose gel slab. and compare the DNA of different individuals. a radioactive complementary DNA probe can be made that will recognize and bind that sequence. the radioactive probe can be used to demonstrate genotypic similarities between individuals. The evidence needed for DNA fingerprinting can be obtained from any biological material that contains DNA: body tissues. whereas a string of four students would require more time and have difficulty working their way through the maze. the rate at which a DNA fragment migrates through the gel is inversely proportional to its size in base pairs. which is placed into a chamber filled with a conductive buffer solution. An individual student can wind his/her way through the maze quickly and with little difficulty. Therefore. These bands will be seen in the gel after the DNA is stained. If a sample of DNA is too small it may be amplified using PCR techniques. etc.Agarose Gel Electrophoresis Agarose gel electrophoresis separates DNA fragments by size. Fragments of the same size stay together and migrate in single bands of DNA. Instructor’s Manual 5 INSTRUCTOR'S MANUAL BACKGROUND . hair follicles. To show that a piece of DNA contains a specific nucleotide sequence. Radioactive probes allow molecular biologists to locate. The size of the fragments reflect variations in individuals’ DNA. In DNA fingerprinting. Over a period of time. smaller DNA fragments will travel farther than larger ones. Since DNA fragments are negatively charged.
The students’ task is to look at the DNA banding patterns and see if any of the suspects’ bands match those of the DNA found at the crime scene.000 bp in a 1% agarose gel. The final reaction buffer consists of 50 mM Tris. comigrates with DNA fragments of approximately 500 bp in a 1% agarose gel. 1 mM DDT. Making DNA Visible DNA is colorless so DNA fragments in the gel cannot be seen during electrophoresis. 6 and 7. 5.base pair (bp)) on a segment of DNA.0. the lanes are numbered from the top left. it cuts the DNA molecule at that point. the stain molecules attach to the DNA trapped in the agarose gel. The DNA from the crime scene has been labeled CS. the “faster” dye. 10 mM MgCl2. The recognition sequences for two commonly used enzymes. while the xylene cyanol. A sample loading buffer containing two bluish dyes is added to the DNA samples. When the gel is immersed in Fast Blast DNA stain. When a particular restriction enzyme "recognizes" a particular recognition sequence (four. one suspect’s DNA is placed in each of lanes 3. pH 8. 4.For the enzyme PstI CTGCAG GACGTC ✄ Like all enzymes. The dye fronts migrate toward the positive end of the gel. the “slower” dye. restriction enzymes are the "chemical scissors" of the molecular biologist. Instructor’s Manual 6 ✄ ✄ INSTRUCTOR'S MANUAL BACKGROUND Restriction Digestion of DNA Because they cut DNA. that from Suspect #1. The proper restriction enzyme buffer has been included with the DNA sample in this kit. The DNA from the crime scene is placed in lane 2. which is the ideal condition for EcoRI and PstI enzymes to function. so that when the rehydrated DNA and enzymes are mixed. 100 mM NaCl. just like the DNA fragments. When the bands are visible. By convention. S1 and so on. EcoRI and PstI. Bromophenol blue. the ideal conditions are created for the enzymes to function optimally. The place on the DNA backbones where the DNA is actually cut is shown with a (✄) symbol: For the enzyme EcoRI G A AT T C C T TA AG ✄ . The loading dye does not stain the DNA itself but makes it easier to load the samples and monitor the progress of the DNA electrophoresis. The gel on page 7 shows the DNA pattern that will be obtained by your students following electrophoresis.or six. Lane 1 contains HindIII lambda digest (DNA size markers). your students can compare the DNA restriction patterns of the different samples of DNA. restriction enzymes function best under specific buffer and temperature conditions. Staining the DNA pinpoints its location on the gel. are shown below. comigrates with DNA fragments of approximately 4.
in analyzing how incriminating the DNA evidence is. In general one can assume that any two humans are 99. But if the frequency of a DNA pattern turning up in a population for a particular segment is extremely low. technicians analyze much larger segments of DNA and many more bands and lanes are produced. 1 in 10?” Instructor’s Manual 7 INSTRUCTOR'S MANUAL BACKGROUND . then very little information will be attained. one needs to ask the question: “Statistically. In actual DNA fingerprinting. some segments will show more variation than others. how many people in a population have the same pattern as that taken from a crime scene: 1 in 1. Depending on demographic factors such as ethnicity or geographic isolation. Therefore.000. It is necessary to examine areas that differ to create a useful DNA fingerprint. then this segment can serve as a powerful tool to discriminate between individuals in that population.M 1 CS 2 S1 3 S2 4 S3 5 S4 6 S5 7 8 It is easy to see that the DNA taken from the crime scene and the DNA from S3 are identical.000? Or. In humans there are thousands of RFLP loci or DNA segments that can be selected and used for fingerprinting analysis. Thus they will differ by only 1 bp in 1. but other evidence may be needed to prove him or her guilty! You may point out to your students that this is a simulation. The degree of variation will affect the statistical odds of more than one individual having the same sequence. Some populations show much less variation in particular DNA segments than others. Different populations show different patterns in their genotypes due to the contributions made to their individual gene pools over time.000.000? 1 in 10.9% identical DNA sequence. Reliability of DNA Evidence Two major factors affecting the reliability of DNA fingerprinting technology in forensics are population genetics and genetic statistics. If 90% of a given population has the same frequency in its DNA fingerprinting pattern for a certain DNA segment. The DNA evidence may place the suspect at the scene. You may want to point out how “strong” or “weak” this evidence is in convicting a suspect.
except that the foreign DNA that was joined to them is also perpetuated. using simple experiments and the use of logic. Scientists routinely take advantage of plasmid DNA because its small size makes it easy to purify. The hybrid plasmids can perpetuate themselves in bacteria just as before.INSTRUCTOR'S MANUAL BACKGROUND Plasmid Mapping Extension The information contained in this kit can be used to perform an optional plasmid mapping activity extension. and the plasmid DNA that carried it is called a “vector”. This antibiotic resistance gave the bacteria harboring these plasmids a selective advantage over their competitors. Plasmids cut with a restriction enzyme can be joined to foreign DNA from any source that has been cut with the same enzyme. Plasmids can be mapped (described) in terms of the location of restriction sites. non-chromosomal pieces of DNA that can replicate in and are commonly found in bacteria and simple eukaryotic organisms such as yeast. We say that the foreign piece of DNA has been “cloned”. In nature. Bacteria were able to pass the beneficial plasmid DNA to other bacteria via conjugation. it is easier and quicker to use a restriction digestion to create a plasmid map. During this hybrid plasmid construction process. The resulting hybrid DNA can then be transformed into bacterial cells. Plasmid mapping is a technique that allows molecular biologists to quickly evaluate the success of cloning experiments and to easily identify plasmids and associated traits in different organisms. Sizes of fragments are then estimated by comparison with known standards. Instructor’s Manual 8 . The sizes of the fragments from the single digests and the double digest are determined then logic is used to assess the relative location of restriction sites. The general procedure is to digest a plasmid with two restriction enzymes separately (two single digests) and then together (a double digest). The crime scene and suspect DNA samples in this kit do not contain human DNA but are constructed using plasmid DNA isolated from bacteria. They typically carry accessory genes separate from the the organism’s genomic DNA. Plasmids are circular. bacteria evolved plasmids containing genes that enabled them to grow in the presence of antibiotics produced by other micro-organisms in the environment. it is necessary to confirm that the foreign DNA has been successfully inserted into the host plasmid. While it is possible to have the complete DNA sequence of each construct determined. Every hybrid plasmid now contains a copy of the piece of foreign DNA joined to it. and once a genetically-engineered DNA sequence has been added it can be reintroduced into bacterial cells using a procedure called transformation.
All activities are designed to be carried out in consecutive 50 minute periods. perform the restriction digests Complete preliminary analysis and review questions Electrophoresis of DNA Samples Load and run gels.Instructor’s Advance Preparation Guide Implementation Timeline There are four activities and two optional extension activities in this fingerprinting curriculum. stain gels Do analysis and review questions Analysis and Interpretation of Results Do analysis questions Generate standard curve Discuss results and weigh evidence Extension Activities Activity 1 Activity 2 Plasmid Mapping Work through questions Constructing a Plasmid Work through questions Instructor’s Manual 9 INSTRUCTOR'S MANUAL ADVANCE PREPARATION . Activities include: • • • A series of prelab considerations for students An active student investigation Questions for analysis and interpretation of lab results Student Schedule Pre-Lab Activity: Activity Lesson 1 Activity Lesson 2 Activity Post-Lab Activity Activity Introduction to DNA Fingerprinting Lecture and discussion Prelab considerations 1 and 2 Restriction Digest of DNA Samples Pour gels.
Activity Read manual Prepare electrophoresis TAE buffer and pour agarose gels When Immediately Prior to or during Lesson 2 Time required 1 hour 1 hour INSTRUCTOR'S MANUAL ADVANCE PREPARATION Rehydrate lyophilized DNA/ buffer samples and enzyme mix and aliquot Prepare Fast Blast DNA stain.Instructor’s Advance Preparation This sections outlines the recommended schedule for advanced preparation on the part of the instructor. A detailed Advance Preparation Guide is provided on pages 13–19. Prepare HindIII standard. Aliquot loading dye and prepared standard Set up workstations Prior to Lesson 2 20 minutes Prior to Lesson 3 20 minutes The day of student labs 10 minutes/day Instructor’s Manual 10 .
and equipment that should be present at a common location. rehydrated 1 vial Suspect 1 (S1) DNA with buffer. red. rehydrated 1 vial Suspect 3 (S3) DNA with buffer. casting tray. you may choose to aliquot stock solutions of DNA and enzymes for the students. is also listed below. The components provided in this kit are sufficient for 8 student workstations (we recommend 2–4 students per workstation). Teacher’s (Common) Workstation. blue. orange. 8-well comb) EcoRI/PstI enzyme mix Pipet tips. rehydrated 1 vial Suspect 4 (S4) DNA with buffer. Materials and supplies that should be present at each student workstation prior to beginning each laboratory experiment are listed below. yellow Permanent marker Waste container Foam micro test tube holder Laboratory tape (not 3M Scotch brand or similar tape) Quantity 1 1 tube (80 µl) 15 tips 1 1 1 1 1 1 ✔ (✔) ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ Instructor’s Workstation Crime scene (C5) DNA with buffer. Instructor’s Manual 11 INSTRUCTOR'S MANUAL ADVANCE PREPARATION .Workstation Checklist Student Workstations. rehydrated 1 vial Suspect 5 (S5) DNA with buffer. 2–20 µl Colored micro test tubes: green. or whether the teacher should aliquot solutions and operate equipment. such as no eating or drinking. A list of materials. should always be practiced. It is up to the discretion of the teacher as to whether students should access common buffer solutions and equipment. Lesson 1 Restriction Digestion of DNA Samples Materials Needed for Each Workstation Agarose gel electrophoresis system (electrophoresis chamber. To avoid the potential for contamination and spills. 2–200 µl Adjustable micropipet. violet. supplies. rehydrated 1 vial Molten 1% agarose in 1x TAE(See Advance Prep page 14) 40–50 ml per gel 37°C water bath or incubator (optional) 1 per class Microcentrifuge 1 per class or mini centrifuge 4 per class ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ Protective eye goggles should be worn in the laboratory at all times. rehydrated 1 vial Suspect 2 (S2) DNA with buffer. which can be accessed by all student groups. All other reagents should be kept at the front of the room for student teams to access as they need them. Proper safety precautions.
2–20 µl Adjustable micropipet. 1x or 100x* Large containers for destaining (if applicable)* Foam micro test tube holder Power supply Gel staining tray Electrophoresis buffer (1x TAE) Instructor’s Workstation Microcentrifuge or mini centrifuge (optional) Rocking platform (optional) Quantity 1 1 6 1 1 1 13 1 1 1 120 ml per 2 stations 1–3 per 2 stations 1 1 1 per 2 stations 275 ml per station ✔ (✔) ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ INSTRUCTOR'S MANUAL ADVANCE PREPARATION 1 4 1 ❑ ❑ ❑ Post-Lab Activity: Analysis of Results Materials Needed for Each Workstation Millimeter ruler Semilog graph paper Gel support film (if applicable)* Quantity 1 1 1 ✔ (✔) ❑ ❑ ❑ Instructor’s Workstation None required * Depending on whether the quick or overnight staining will be followed. Instructor’s Manual 12 . 2–20 µl Waste container Gel support film (if applicable)* Fast Blast DNA stain.Lesson 2 Electrophoresis of DNA Samples Materials Needed for Each Workstation Agarose gel electrophoresis system Agarose gel Digested DNA samples HindIII lambda digest (DNA marker) DNA sample loading dye Permanent marker Pipet tips.
It is critical that the enzyme mix is kept on ice. prepare the molten agarose ahead of time. and combs Electrophoresis buffer (50x TAE) Agarose powder 8 clear microtubes 3 liters distilled water Time required: What’s required: Procedures Note: All of the DNA and enzyme vials should contain a white residue. 100 mM NaCl. Replace the stopper and vigorously shake the vial. 2 mM DTT. pH 8. casting trays. the final concentration of buffer will be 50 mM Tris. An estimation of preparation time is included in each section. Transfer 80 µl of the rehydrated enzyme mix into each of eight. Aliquot enzyme mix. 1 mM DTT. If prepared in advance.5 ml microtubes to make pipetting easier for your students. The rehydrated enzymes should be used within 12 hours. Rehydrate DNA samples A. Allow DNA/buffer samples to rehydrate at room temperature for 15 minutes or until dissolved. Set temperature of 37°C for water bath or incubator Set up student and instructor workstations Thirty minutes to 1 hour.0. 200 mM NaCl. Instructor’s Manual 13 INSTRUCTOR'S MANUAL ADVANCE PREPARATION . Lesson 1 Restriction Digestion of DNA Samples Advance Preparation Objectives: Rehydrate DNA/buffer samples and restriction enzymes Aliquot restriction enzymes Pour agarose gels to prepare for lesson 2. labeled 1. molten agarose should be kept in a water bath set at 50–55°C until gels are poured. 10 mM MgCl2. It is critical to dissolve all the powder. If you prefer to have your students pour their own gels during the lab. Rehydrate lyophilized EcoRI/PstI enzyme mix. The lyophilized DNA samples have color-coded labels on clear glass vials. which may appear as a loose powder in the DNA vials.0. To rehydrate DNA samples.Instructor’s Advance Preparation for Labs This section describes the preparation that needs to be performed by the instructor before each laboratory. Gentle heating at 37°C for 10 minutes may be necessary. which is the ideal condition for EcoRI and PstI enzymes to function. carefully remove the stopper and add 200 µl of sterile water to each lyophilized DNA vial. but not frozen. 2. add 750 µl sterile water and swirl to resuspend the enzymes. The rehydrated DNA samples are now at a concentration of 0.5 ml microtubes labeled ENZ. The lyophilized EcoRI/PstI enzyme mix is in an amber vial. 3. clear 1. pH 8.3 µg/µl in 100 mM Tris. 1. To rehydrate EcoRI/PstI enzyme mix. You may choose to transfer the rehydrated DNA/buffer samples to color-coded. once it has been rehydrated. Allow enzymes to rehydrate on ice for 5 minutes. depending on how you choose to prepare agarose gels Horizontal electrophoresis gel chamber. Once the DNA in buffer is added to the enzyme. 20 mM MgCl2. some of which may be stuck to the stopper.
Boil and swirl the solution until all of the small transparent agarose particles are dissolved. If using an Erlenmeyer flask. invert a 25 ml flask into the open end of the 500 ml Erlenmeyer flask containing the agarose.g. not water. Volume of 1% agarose for: Number of gels 7 x 7 cm tray 7 x 10 cm tray 1 2 4 8 40 ml 80 ml 160 ml 320 ml 50 ml 100 ml 200 ml 400 ml INSTRUCTOR'S MANUAL ADVANCE PREPARATION b. Caution: Always wear protective gloves. add 60 ml of 50x concentrate to 2. The agarose can be melted for gel casting by boiling until agarose has melted completely on a magnetic hot plate. and lab coat while preparing and casting agarose gels. Agarose preparation. This technique is the fastest and safest way to dissolve agarose. Be sure to use electrophoresis buffer. Use this table as a guide for gel volume requirements when casting single or multiple gels. In addition to the 1x TAE buffer needed to make the agarose gels.75–1. Boiling molten agarose or the flasks containing hot agarose can cause severe burns if allowed to contact skin. Add the appropriate amount of 1x TAE electrophoresis buffer and swirl to suspend the agarose powder in the buffer.0 cm for easy sample loading and gel handling. Add the agarose powder to a suitable container (e. 1% TAE gels and fit into Bio-Rad’s Mini-Sub Cell GT cell or any horizontal gel electrophoresis system that fits 7 x 10 cm gels. The recommended thickness for the gel is 0. ii. to prepare agarose gels. hot water bath. These procedures may be carried out 1 to 2 days ahead of time by the teacher or done during class by the individual student teams. Be sure to use electrophoresis buffer. Microwave oven method. If electrophoresis chambers are limiting.* The recommended agarose concentration for gels in this classroom application is 1% agarose. Instructor’s Manual 14 .4. Electrophoresis buffer preparation. 500 ml Erlenmeyer flask for 200 ml or less). Three liters of 1x TAE buffer will be sufficient to run 8 electrophoresis chambers and prepare 8 agarose gels. (See Appendix D for alternative Fast Gel Protocol) a. Stop the microwave oven every 30 seconds and swirl the flask to suspend any undissolved agarose. The small flask acts as a reflux chamber. These are 2 x 8-well. To make 3 L of 1x TAE from 50x TAE concentrate. This concentration of agarose provides good resolution and minimizes run time required for electrophoretic separation of DNA fragments. you can use a 7 x 10 cm tray and two 8-well combs to pour a gel that can be used to run two sets of student digests. use 1 gram of agarose for each 100 ml of 1x TAE electrophoresis buffer. Place the gel solution in an appropriate bottle or flask into the microwave. * Convenient precast agarose gels (catalog #161-3057EDU) are available from Bio-Rad. TAE (Tris-acetate-EDTA) electrophoresis buffer is provided as a 50x concentrated solution. thus allowing long or vigorous boiling without much evaporation. or in a microwave oven. goggles. not water. Use a medium setting and set to 3 minutes. To make a 1% agarose solution. approximately 275 ml is also required for each electrophoresis chamber. LOOSEN THE CAP IF YOU ARE USING A BOTTLE. i. Prepare agarose gels.94 L of distilled water.. Heated agarose can quickly boil over so watch the process carefully. Set aside and cool to 55–60°C before pouring.
Level the gel tray on a leveling table or workbench using the leveling bubble provided with the chamber. Procedure for Casting Gels This laboratory activity requires that each gel has at least 7 wells. Seal the ends of the gel tray securely with strips of standard laboratory tape (not Scotch tape or similar). floating them in a large volume (1 liter or more) of 37°C water. The combs will form the wells into which the samples will be loaded. or opaque. The time needed to pour gels by an entire class is approximately 30 minutes. and allowing them to incubate overnight as the water cools to room temperature. 15 INSTRUCTOR'S MANUAL ADVANCE PREPARATION . vi. This section outlines the tape-the-tray method for casting gels. store gels in a sealable plastic bag with 1–2 ml of 1x TAE running buffer at room temperature for 1 day or in the refrigerator (4°C) for up to 1 week before using. Have your students label their plastic bags.5–0. when ready to use. Boil the solution until all of the small transparent agarose particles are dissolved. Press the tape firmly to the edges of the gel tray to form a fluid-tight seal. Bubbles or foam should be disrupted before rising to the neck of the flask. Set aside to cool to 55–60°C before pouring gels. Add a stirbar to the undissolved agarose solution. Have students label their plastic bags. place the tray onto the leveled DNA electrophoresis chamber so that the sample wells are at the black (cathode) end of the base. pour one or two extra gels for back-up. Restriction Digests. Heat the solution to boiling while stirring on a magnetic hot plate. Pour enough agarose to cover the gel comb teeth or to a depth of 0. Carefully remove the comb from the solidified gel. place one comb at one end of the tray and the other comb in the middle of the tray. Follow the instructions above to prepare the agarose and to determine what volume of 1% agarose will be needed for your class(es). Instructor’s Manual i. iii. While the agarose is cooling to 60°C. vii. ii. viii. Option 2: If there is sufficient time to proceed to Lesson 2. If a 37°C heating block. iv. place the comb into the appropriate slot of the gel tray. Other methods are detailed in the Sub-Cell GT cell (electrophoresis chamber) instruction manual. DNA samples will migrate towards the red (anode) end of the chamber during electrophoresis. samples can be digested by placing tubes in foam micro test tube holders.75 cm. You have two options: Option 1: If you do not have sufficient time to proceed to Lesson 2. A 45-minute incubation at 37°C is the optimum digestion condition. 7 x 7 cm gel is cast. v. ix. It will appear cloudy. Allow the gel to solidify at room temperature for 10 to 20 minutes. Solidified gels can be stored in sealable bags at room temperature for 1 day or in the refrigerator for up to 1 week before using.Magnetic hot plate method. Do not move or handle the gel tray until the gel has solidified. Prepare the desired concentration and amount of agarose in 1x TAE electrophoresis buffer. If possible. To pour a double-well gel using a 7 x 10 cm tray and two 8-well combs. Remove the tape from the edges of the gel tray. water bath or incubator is not available. Gel combs should be placed within 1/2 inch of the end of the gel casting tray if a single-well. c. Cool the agarose to at least 60°C before pouring.
Here is a quick summary on how to use micropipets: 1. Eject the pipet tip. Insert the pipet tip into the solution to be transferred. Press the plunger past the first stop to the second (hard) stop to transfer the liquid. INSTRUCTOR'S MANUAL ADVANCE PREPARATION 4. 6. Twist the dial on the micropipet to set the desired volume. Insert the pipet tip into the desired tube. Look at the micropipet to determine the volume range. Slowly release the plunger to retrieve the liquid. 7.Practice Using Micropipets (Optional) We recommend that you familiarize your students with proper pipeting techniques prior to Lesson 1. Instructor’s Manual 16 . 5. 9. 8. 3. 2. Attach a clean pipet tip. Students may practice by using either sample loading dye or food coloring mixed with either a dense saturated sugar or glycerol solution. Make sure to keep the plunger pressed when lifting the pipet tip out of the tube. Press the micropipet plunger to the first (soft) stop. Have your students learn how to transfer different volumes of a solution from one tube into another with a micropipet.
Instructor’s Manual 17 INSTRUCTOR'S MANUAL ADVANCE PREPARATION Time required 45 minutes . Refer to Appendix D for detailed information. if you have not prepared it already. 2. b. then chill on ice — this results in better separation of the marker bands. Heat the marker to 65°C for 5 minutes. See laboratory protocol in the student section. casting trays.25 x TAE buffer is used for fast gel electrophoresis. Prepare Fast Blast DNA stain a. sample loading and electrophoresis can begin. and on type of electrophoresis buffer used. and combs Electrophoresis buffer (1x TAE)* Fast Blast DNA stain. Note: Power requirements vary depending on gel thickness. Submerge the gel under about 2 mm of 1x TAE buffer. DNA samples will migrate toward the red anode end during electrophoresis. For this exercise we recommend using a constant voltage of 100 V for 30 min. Label clear micro test tubes “M”. 4. and concentration. dilute 1 ml of 500x Fast Blast with 499 ml of distilled or deionized water in an appropriately sized flask or bottle and mix.Lesson 2 Agarose Gel Electrophoresis and Visualization of DNA Fragments Advance Preparation Objectives Prepare HindIII lambda digest (DNA marker) and aliquot (optional) Aliquot sample DNA loading dye (optional) Prepare the electrophoresis chamber Dilute Fast Blast DNA strain to 1x (for overnight staining) or 100x concentration (for quick staining) Set up student and teacher workstations What is required HindIII lambda digest (DNA marker) Sample loading dye Electrophoresis chambers. Prepare the required volume of 1x TAE buffer. Distribute one tube to each team. Make sure the tray is fully seated in the chamber. When the agarose gel has solidified. Prepare the electrophoresis chamber. Prepare samples for gel loading. When placing the gel tray into the electrophoresis chamber. Prepare HindIII lambda digest (DNA marker) and aliquot (optional). dilute 100 ml of 500x Fast Blast with 400 ml of distilled or deionized water in an appropriately sized flask or bottle and mix. Cover the flask and store at room temperature until ready to use. To prepare 1x stain (for overnight staining). d. length. a. Aliquot DNA sample loading dye. To prepare 100x stain (for quick staining). Add 20 µl of DNA sample loading dye to the stock tube containing the HindIII lambda DNA marker. 500x Procedures 1. b. 3. Label eight clean micro test tubes “LD” for loading dye and aliquot 50 µl of sample loading dye into each tube. c. * 0. See Appendix D for a faster electrophoresis protocol which allows the gel to be run in 20 min. Cover the flask and store at room temperature until ready to use. Aliquot 15 µl of the DNA markers containing loading dye to 8 clear micro test tubes labeled “M”. make sure that the sample wells are at the black cathode end.
Each student team may utilize separate washing containers for each wash step. Detailed instructions on using Fast Blast are included in the student manual. The proprietary dye formula stains DNA deep blue in agarose gels and provides vivid. agarose gels must be removed from their gel trays before being placed in the staining solution. Dispose of the staining solutions according to protocols at your facility. add a sufficient volume of staining solution to completely submerge the gels. We highly recommend using a large spatula or other supportive surface to transfer the gel from one container to another during the destaining steps involved with the quick staining protocol. and nontoxic alternative to ethidium bromide for the detection of DNA in agarose gels following electrophoresis. latex or vinyl gloves should be worn while handling the stain or stained gels to keep hands from becoming stained blue. • • • • • Instructor’s Manual 18 . When the DNA bands are visible. Fast Blast contains a cationic compound that is in the thiazin family of dyes. The stain can be used as a quick stain when diluted to a 100x concentration to allow the visualization of DNA within 12–15 minutes or used as an overnight stain when diluted to 1x concentration. The positively charged dye molecules are attracted to and bind to the negatively charged phosphate groups on DNA molecules. special attention must be given when handling it. Verify that these solutions do not harm the surface prior to use. or simply use a single container that is emptied after each wash and refilled for the next wash. at each student workstation. consistent results and detects as little as 50 nanograms of DNA. INSTRUCTOR'S MANUAL ADVANCE PREPARATION WARNING Although Fast Blast DNA stain is nontoxic and noncarcinogenic. Because the gel is fragile. capable of holding at least 500 ml. If alternative staining trays are used. Use either 10% bleach solution or a 70% alcohol solution to remove Fast Blast from most surfaces. Lab coats or other protective clothing should be worn to avoid staining clothes. your students can compare the DNA restriction patterns of the different samples of DNA. the dye molecules attach to the DNA molecules trapped in the agarose gel. Note: • We recommend using 120 ml of diluted Fast Blast to stain two 7 x 7 cm or 7 x 10 cm agarose gels in individual staining trays provided in the kit (you may want to notch gel corners for identification). Destaining (when performing the quick staining protocol) requires the use of at least one large-volume container. No washing or destaining is required when using the overnight staining protocol. safe. Fast Blast DNA stain is provided as a 500x concentrate that must be diluted prior to use.Making DNA Visible Fast Blast DNA stain is a convenient. This is easily accomplished by holding the base of the gel tray in one hand and gently pushing out the gel with the thumb of the other hand. Following electrophoresis. 100x Fast Blast can be reused at least seven times. When the agarose gel is immersed in Fast Blast DNA stain.
Graphing the Data Many of your students may not be familiar with logarithms and semilog graph paper. As the gel dries. Gel Support Film Note: Avoid extended exposure of dried gels to direct light to prevent band fading. Other gel media may not be appropriate for this purpose. Instructor’s Manual 19 INSTRUCTOR'S MANUAL ADVANCE PREPARATION . the gel will dry completely at room temperature after 2–3 days. Drying the Agarose Gel as a Permanent Record of the Experiment Note: Drying agarose gels requires the use of Bio-Rad’s specially formulated high-strength analytical grade agarose. Place the gel directly upon the hydrophilic side of a piece of gel support film. making sure to avoid direct exposure to light. You might also choose to discuss the advantage of using semilog vs. it will bond to the film but will not shrink. The result will be a flat. either trace the gel outline (including wells and DNA bands) on a piece of paper or acetate. Remove the stained agarose gel from its staining tray and trim away any unloaded lanes with a knife or razor blade. transparent.To obtain a permanent record of the gel before it is dried.) Center the gel on the film and remove bubbles that may form between the gel and film. standard graph paper in this instance. We have included semilog graph paper on page 44 of this manual. A math extension here can also provide an opportunity to explore linear and exponential (arithmetic and geometric) sequences of numbers. However. take a photograph with a digital camera. (Water will form beads on the hydrophobic side but will spread flat on the hydrophilic side of the film. and durable record of the experiment. Place the film on a paper towel and let the gel dry. It is suggested that you prepare a short lesson to demonstrate the proper way to label the coordinates and plot the points. We recommend using Bio-Rad’s exclusive gel support film (catalog #170-2984EDU) to dry agarose gels. DNA bands will reappear if the dried gels are stored in the dark for 2–3 weeks after fading. photocopy the gel or scan the gel. If left undisturbed on the support film.
Place the tubes in the foam micro test tube holder. Use a fresh tip to transfer the ENZ sample to each tube. Pipet 10 µl of enzyme mix (ENZ) into the very bottom of each tube. Otherwise. Tap 7. Stock CS S1 S2 S3 S4 S5 5. proceed directly to step 2 of Lesson 2. on ice.Forensic DNA Fingerprinting Kit Quick Guide Lesson 1 Restriction Digestion 1. 20 Student Manual . Tightly cap the tubes and mix the components by gently flicking the tubes with your finger. date. gently tap the tube on the table top. If a microcentrifuge is available. Flick 6. Ice CS S1 S2 S3 S4 S5 3. Place the tube containing the restriction enzyme mix. and lab period. After the incubation period. Label one of each colored micro test tubes as follows: green tube CS (crime scene) blue tube S1 (suspect 1) orange tube S2 (suspect 2) violet tube S3 (suspect 3) red tube S4 (suspect 4) yellow tube S5 (suspect 5) Label the tubes with your name. DNA Samples + Enzyme Mix QUICK GUIDE 4. follow the instructors directions to pour a 1% agarose gel. Make sure the sample is transferred to the bottom of the tubes. pipet 10 µl of each DNA sample from the stock tubes and transfer to the corresponding colored micro test tubes. Using a fresh tip for each sample. If required. Pipet up and down carefully to mix well. ENZ 2. Water bath 8. labeled ENZ. If there is sufficient time to continue. Place the tubes in the foam micro tube holder and incubate for 45 min at 37°C or overnight at room temperature in a large volume of water heated to 37°C. pulsespin in the centrifuge to collect all the liquid in the bottom of the tube. remove the tubes from the water bath and place in the refrigerator until the next laboratory period.
Cap the tubes and mix by gently flicking the tube with your finger. blue tube. Using a separate tip for each sample. 20 µl 8. 3. add 5 µl of loading dye "LD" into each tube. 20 µl Lane 6: S4. Plug the electrodes into the power supply. Remove the agarose gel from the refrigerator (if applicable) and remove the plastic wrap. red to red and black to black. load the indicated volume of each sample into 7 wells of the gel in the following order: Lane 1: M. Collect the sample at the bottom of the tube by tapping it gently on the table or by pulse-spinning in a centrifuge. DNA size marker. red tube. pulse spin the tubes in the centrifuge to bring all of the liquid into the bottom of the tube or gently tap on the table top. orange tube.Lesson 2 Agarose Gel Electrophoresis 1. Remove the digested DNA samples from the refrigerator (if applicable). 20 µl Lane 5: S3.25x TAE if using the Fast Gel Protocol Student Manual DNA Loading Dye (+) (–) 21 QUICK GUIDE . If a centrifuge is available. 6. 20 µl Lane 7: S5. Turn on the power and electrophorese your samples at 100 V for 30 minutes. Place the agarose gel in the electrophoresis apparatus. green tube. Check that the wells of the agarose gels are near the black (–) electrode and the bottom edge of the gel is near the red (+ ) electrode. 9. 10 µl Lane 2: CS. Using a separate tip for each sample. horizontal electrophoresis chamber. 5. 7. Carefully place the lid on the electrophoresis chamber. Centrifuge 2. 4. 20 µl Lane 4: S2. The red and black jacks on the lid of the horizontal electrophoresis chambers will match with the red and black jacks on the base. Fill the electrophoresis chamber with 1x TAE buffer* to cover the gel. 20 µl Lane 3: S1. The lid will attach to the base in only one orientation. yellow tube. violet tube. * or 0. using approximately 275 ml of buffer for a Bio-Rad Mini-Sub Cell.
QUICK GUIDE Student Manual 22 . Overnight staining a. Pour off the stain into a waste beaker. Add 120 ml of 100x Fast Blast DNA stain into a staining tray (2 gels per tray). c. d. d. Let the gels stain overnight. b. e. Trim away any unloaded lanes. Record results. e. Stain the gels for 2 minutes with gentle agitation. with gentle shaking for best results. f. Carefully remove the gel and tray from the gel box. Add 120 ml of 1x Fast Blast DNA stain to the staining tray (2 gels per tray). Air-dry the gel on gel support film and tape the dried gel into your laboratory notebook. Save the used stain for future use. Slide the gel into the staining tray. c.Visualization of DNA Fragments 1. No destaining is required. Air-dry the gel on gel support film and tape the dried gel into your laboratory notebook. Trim away any unloaded lanes. f. Be careful — the gel is very slippery. turn off the power and remove the top of the chamber. 2. Record results. Destain by washing twice in warm tap water for 5 minutes each with gentle shaking for best results. b. When the electrophoresis run is complete. Transfer the gels into a large washing container and rinse with warm (40–55°C) tap water for approximately 10 seconds. g. You have two options for staining your gel: Quick staining (requires 12–15 minutes) a.
These base pairs are in turn bonded to a sugar-phosphate backbone. guanine. Student Manual 23 STUDENT MANUAL BACKGROUND . The Structure of DNA The schematics above represent a very small section of DNA from three different individuals. G. thymine. Remember the base-pairing rule is A . Refer to the figure below of a DNA molecule.C. and cytosine (A. The four nitrogenous bases are adenine. T.T and G . and C).Student Manual Pre-Lab Introduction to DNA Fingerprinting You are about to perform a procedure known as DNA fingerprinting. DNA consists of a series of nitrogenous base molecules held together by weak hydrogen bonds. For this experiment it is necessary to review the structure of DNA molecules. The data obtained may allow you to determine if the samples of DNA that you will be provided with are from the same individual or from different individuals. In this representation of DNA the symbol system is as follows: Backbone: S = Five carbon sugar molecule known as deoxyribose P = Phosphate group DNA Nucleotide Bases: A = adenine C = cytosine G = guanine T = thymine Analysis of the three DNA samples above (see next page) might help us detect similarities and differences in samples of DNA from different people.
what conclusions can you make about the similarities and differences of the DNA samples? 5. In the above figure.STUDENT MANUAL PRE-LAB ACTIVITY Pre-Lab Focus Questions: Introduction to DNA Fingerprinting Consideration What is the structure of DNA? 1. What will you need to compare between these DNA samples to determine if they are identical or non-identical? Student Manual 24 . 4. Are the bases paired in an identical manner in all three samples? Describe the pattern of the base pair bonding. Compare the “backbone” of the sugar-phosphate arrangement in the side chains of all three figures. In your attempt to analyze DNA samples from three different individuals. 3. Are there any differences? 2. do all three samples contain the same bases? Describe your observations.
Left: Right: 3. You need to determine if the linear base pair sequence in the DNA samples is identical or not! An understanding of some historically important discoveries in recombinant DNA technology might help you to develop a plan. How many pieces of DNA would result from this cut? ___________ 2. These molecular scissors or “cutting” enzymes are restriction endonucleases. first you must understand and visualize the nature of the "cutting" effect of a restriction endonuclease on DNA: AT G A AT T C T C A AT TAC C T TAC T TA AG AG T TA AT G G A The line through the base pairs represents the sites where bonds will break if the restriction endonuclease EcoRI recognizes the site GAATTC. In 1968. The following analysis questions refer to how a piece of DNA would be affected if a restriction endonuclease were to "cut" the DNA molecule in the manner shown above. Write the base sequence of the DNA fragments on both the left and right side of the “cut”. 1. Switzerland and Dr. your task might seem rather difficult. This causes the double strand of DNA to break along the recognition site and the DNA molecule becomes fractured into two pieces. discovered a group of enzymes in bacteria. Dr. Hamilton Smith at the Johns Hopkins University. Two common restriction enzymes (endonucleases) are EcoRI and PstI which will be provided to you in this lab procedure. What differences are there in the two pieces? ✄ Student Manual 25 STUDENT MANUAL LESSON 1 ✄ . To better understand how EcoRI and PstI may help you in performing your DNA fingerprinting experiment. which when added to any DNA will result in the breakage [hydrolysis] of the sugar-phosphate bond between certain specific nucleotide bases [recognition sites]. Werner Arber at the University of Basel. Baltimore.Lesson 1 Restriction Digestion of DNA Samples Consideration How can we detect differences in base sequences? At first sight.
single strands are shown for simplicity: Sample #1 STUDENT MANUAL LESSON 1 CAGTGATCTCGAATTCGCTAGTAACGTT Sample #2 TCATGAATTCCTGGAATCAGCAAATGCA If both samples are treated with the restriction enzyme EcoRI [recognition sequence GAATTC] then indicate the number of fragments and the size of each fragment from each sample of DNA. Indicate the size of the fragments [mention any discrepancy you may detect]. DNA fragment size can be expressed as the number of base pairs in the fragment.4. b) What is the length of the longer fragment? ______________ 5. Consider the two samples of DNA shown below . a) The smaller fragment is ___________ base pairs (bp). Sample # 1 Sample # 2 # of fragments:________ # of fragments:_________ List fragment size in order: largest ——> smallest Sample # 1 Sample # 2 Student Manual 26 .
it is apparent that the only difference between the DNA of different individuals is the linear sequence of their base pairs. How restriction endonucleases cut (hydrolyze) DNA molecules. rehydrated Suspect 2 DNA with buffer. casting tray. yellow Permanent marker Waste container Foam micro test tube holder Laboratory tape (not 3M Scotch brand or similar tape) Instructor’s Workstation Crime scene DNA with buffer. red. rehydrated Suspect 4 DNA with buffer. blue. orange. Your Workstation Checklist Make sure the materials listed below are present at your lab station prior to beginning the lab. rehydrated Suspect 3 DNA with buffer. Now that you have a fairly clear understanding of these three items you are ready to proceed to the first phase of the DNA fingerprinting procedure—performing a restriction digest of your DNA samples.Lesson 1 Restriction Digestion of DNA Samples Upon careful observation. How adding the same restriction endonuclease to two samples of DNA might provide some clues about differences in their linear base pair sequence. 2–200 µl Micropipet. rehydrated Suspect 1 DNA with buffer. Material Needed for Each Workstation Agarose gel electrophoresis system (electrophoresis chamber. 8-well comb) EcoRI/PstI enzyme mix Pipet tips. 2–20 µl Colored micro test tubes: green. rehydrated Molten 1% agarose in 1x TAE (See Advance Prep) 37°C water bath or incubator (optional) Microcentrifuge or mini centrifuge Quantity 1 1 tube (80 µl) 15 tips 1 1 1 1 1 1 ✔ (✔) ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ 1 vial 1 vial 1 vial 1 vial 1 vial 1 vial 40–50 ml per gel 1 per class 1 per class 4 per class ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ Student Manual 27 STUDENT MANUAL LESSON 1 . violet. In the lab. Thus far you have learned the following: • • • The similarities and differences between the DNA from different individuals. Recall that your task is to determine if any of them came from the same individual or if they came from different individuals. rehydrated Suspect 5 DNA with buffer. your team will be given 6 DNA samples.
Using a new pipet tip for each sample. Note: Change tips whenever you switch reagents. change the tip! DNA goes in the tube before the enzyme. 2) Is there any observable difference between the samples of DNA? 3) Describe the appearance of the restriction endonuclease mix. Pipet up and down carefully to mix well. Always add the enzyme last. pipet 10 µl of the enzyme mix “ENZ” to each reaction tube as shown below. 4) Combine and react.Observations 1) Describe the samples of DNA (physical properties). if the tip touches any of the liquid in one of the tubes accidentally. STUDENT MANUAL LESSON 1 ENZ CS S1 S2 S3 S4 S5 Now your DNA samples should contain: DNA Samples (10 µl each) Crime Scene [CS] Suspect 1 [S1] Suspect 2 [S2] Suspect 3 [S3] Suspect 4 [S4] Suspect 5 [S5] EcoRI/PstI Enzyme Mix 10 µl 10 µl 10 µl 10 µl 10 µl 10 µl Total Reaction Volume 20 µl 20 µl 20 µl 20 µl 20 µl 20 µl Student Manual 28 . or. When in doubt.
Mix the tube contents. Tapping the tubes on the lab bench will also help to combine and mix the contents. briskly shake the tube (once is sufficient) like a thermometer. Alternatively. If your lab is not equipped with a centrifuge. (Be sure the tubes are in a BALANCED arrangement in the rotor). After the incubation. Water bath Note: While you are waiting. store the DNA digests in the refrigerator until the next lab period. Mix the components by gently flicking the tubes with your finger. Place the tubes in the foam micro tube holder and incubate them at 37°C for 45 minutes.5. unless they have already been prepared for you. Incubate the samples. or proceed directly to step 2 of Lesson 2 if instructed by your teacher. this is a good time to cast your agarose gel. Student Manual 29 STUDENT MANUAL LESSON 1 . Check with your teacher for the proper procedure. Tightly cap on each tube. pulse the tubes for two seconds to force the liquid into the bottom of the tube to mix and combine reactants. the tubes can be incubated in a large volume of water heated to 37°C and allowed to slowly reach room temperature overnight. CS S1 S2 S3 S4 S5 Flick Tap 6. If there is a centrifuge available.
Lesson 1 Restriction Digestion of DNA Samples Review Questions 1. Before you incubated your samples. 3. is it still possible that the DNA samples were fragmented? Explain your reasoning. describe any visible signs of change in the contents of the tubes containing the DNA after it was combined with the restriction enzymes. Can you see any evidence to indicate that your samples of DNA were fragmented or altered in any way by the addition of EcoRI/PstI? Explain. Student Manual 30 . 4. are there any visible clues that the restriction enzymes may have in some way changed the DNA in any of the tubes? Explain your reasoning. In the absence of any visible evidence of change. (Answer the next day—after the restriction digest) After a 24 hour incubation period. STUDENT MANUAL LESSON 1 2.
.Lesson 2 Agarose Gel Electrophoresis (Laboratory Procedure) Student Workstations Agarose gel electrophoresis system Agarose gel Digested DNA samples HindIII lambda digest (DNA markers) DNA sample loading dye Permanent marker Pipet tips. 2–20 µl Micropipet. Refer to Appendix D for detailed information. 2–20 µl Waste container Gel support film (if applicable)* Fast Blast DNA stain. 1x or 100x* Large containers for destaining (if applicable)* Foam micro test tube holder Power supply Gel staining tray Electrophoresis buffer (1x TAE)** Instructor’s Workstation Microcentrifuge or mini centrifuge (optional) Rocking platform (optional) *If performing the quick staining procedure.25 x TAE buffer is used for fast gel electrophoresis. Quantity 1 1 6 1 1 1 13 1 1 1 120 ml per 2 stations 1–3 per 2 stations 1 1 1 per 2 stations 275 ml per station ✔ (✔) ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ ❑ 1 4 1 ❑ ❑ ❑ Student Manual 31 STUDENT MANUAL LESSON 2 ** 0.
The loading dye should already have been added by your instructor. After preparing the gel. remove the comb from the gel by pulling it straight up. or if your teacher instructs you to do so. 32 Student Manual . Pour buffer in the gel box until it just covers the wells of the gel by 1–2 mm. remove your digested samples from the refrigerator. prepare your own gel.Lesson 2 Agarose Gel Electrophoresis (Laboratory Procedure) 1. where the black lead is connected. If a centrifuge is available. 2. Using a new tip for each sample add 5 µl of sample loading dye "LD" to each tube: DNA Samples Crime Scene [CS] Suspect 1 [S1] Suspect 2 [S2] Suspect 3 [S3] Suspect 4 [S4] Suspect 5 [S5] Loading Dye Loading dye 5 µl 5 µl 5 µl 5 µl 5 µl 5 µl CS LD S1 S2 S3 S4 S5 Flick Tap STUDENT MANUAL LESSON 2 Tightly cap each tube. – + 5. into the platform in the gel box. The wells should be at the (–) cathode end of the box. gently tap the tubes on the table top. pulse spin the tubes to bring the contents to the bottom of the tube. Place the casting tray with the solidified gel in it. Obtain a prepoured agarose gel from your teacher. Very carefully. 4. 3. Obtain the tube of HindIII lambda digest (DNA marker). Otherwise. Pour ~ 275 ml of electrophoresis buffer into the electrophoresis chamber. Mix the components by gently flicking the tubes with your finger.
20 µl S2. + While you are waiting for the gel to run. Connect electrical leads to the power supply. When the electrophoresis is complete. Using a separate pipet tip for each sample. The first sample is loaded in the well at the left hand corner of the gel. 20 µl S3. blue tube. you may begin the review questions on the following page. The gel can be run for up to 40 min to improve resolution if the time is available. Set it for 100 V and electrophorese the samples for at least 30 min. 20 µl 7. green tube. Be careful. Secure the lid on the gel box. Student Manual 33 STUDENT MANUAL LESSON 2 . orange tube. 20 µl S4. 9.6. the gel is very slippery! Proceed to pg 35 for detailed instructions on staining your gel. 20 µl S1. Lane 1: Lane 2: Lane 3: Lane 4: Lane 5: Lane 6: Lane 7: HindIII DNA size marker. 20 µl S5. clear tube. 8. Gels are read from left to right. yellow tube. red tube. The lid will attach to the base in only one orientation: red to red and black to black. turn off the power supply and remove the lid from the gel box. violet tube. Turn on the power supply. Carefully remove the gel tray and the gel from the electrophoresis chamber. 10 µl CS. The Fast Gel Protocol in Appendix D allows the gel to be run in 20 min at 200 V. load your digested DNA samples into the gel.
DNA molecules are negatively charged. small) would you expect to move toward the opposite end of the gel most quickly? Explain. Student Manual 34 . small) are expected to travel the shortest distance from the well? Explain.Lesson 2 Agarose Gel Electrophoresis Review Questions 1. they are “forced” to move through the gel matrix. The electrophoresis apparatus creates an electrical field with positive and negative poles at the ends of the gel. After DNA samples are loaded into the sample wells. What size fragments (large vs. 2. STUDENT MANUAL LESSON 2 4. Which fragments (large vs. To which electrode pole of the electrophoresis field would you expect DNA to migrate? (+ or -)? Explain. What color represents the negative pole? 3.
. Analyze the number and positions of visible DNA bands on your gel. These blue stain molecules strongly bind to the DNA fragments and allow DNA to become visible. it is not immediately visible in the gel. DNA fragments are visualized by staining the gel with a blue stain called Fast Blast DNA stain. Making DNA Fragments Visible Since DNA is naturally colorless. These visible bands of DNA may then be traced. Note that this picture is an example and it may not correspond to the pattern of bands that you will see in the lab.Staining DNA with Fast Blast DNA Stain (Laboratory Procedure) Consideration Are any of the DNA samples from the suspects the same as that of the individual at the crime scene? Take a moment to think about how you will perform the analysis of your gel. photographed. Visualize DNA fragments in your gel. the following information is important to remember: • • Each lane has a different sample of DNA Each DNA sample was treated with the same restriction endonucleases. or retained as a permanently dried gel for analysis. B. then answer the questions on page 40. In the final two steps. Unaided visual examination of the gel after electrophoresis indicates only the positions of the loading dyes and not the positions of the DNA fragments. sketched. The blue stain molecules are positively charged and have a high affinity for the DNA. you will: A. The drawing below represents an example of a stained DNA gel after electrophoresis. For fingerprinting analysis. analyze the bands in the gel drawing below. Detained instruction on staining your gel are found on the following pages. Lane 1 2 3 4 5 6 Student Manual 35 STUDENT MANUAL LESSON 2 With reference to the numbered lanes.
latex or vinyl gloves should be worn while handling the stain or stained gels to keep hands from becoming stained blue. Pour approximately 120 ml of 100x stain into the staining tray. warm (40–55°C) tap water. Because the gel is fragile. Stain the gels for 2–3 minutes. If alternative staining trays are used. and option 2 for overnight staining. but not for more than 3 minutes. capable of holding at least 500 ml. add more 100x stain to completely submerge the gels. Label the staining trays with your initials and class period. Following electrophoresis. agarose gels must be removed from their gel trays before being placed in the staining solution. Stain gels Remove each gel from the gel tray and carefully slide it into the staining tray. You will stain 2 gels per tray. If necessary. Two student teams will stain the gels per staining tray (you may want to notch gel corners for identification). or simply use a single container that is emptied after each wash and refilled for the next wash. For quick staining. Gently shake the gel in the water for ~10 seconds to rinse. 2. Destaining requires the use of at least one large-volume container. Use option 1 for quick staining of gels to visualize DNA bands in 12–15 minutes. Mark staining trays with initials and class period before beginning this activity. Protocol 1: Quick Staining of Agarose Gels in 100x Fast Blast DNA Stain This protocol allows quick visualization of DNA bands in agarose gels within 15 minutes. Depending on the amount of time available. your teacher will decide which protocol to use. Lab coats or other protective clothing should be worn to avoid staining clothes. WARNING Although Fast Blast DNA stain is nontoxic and noncarcinogenic. add a sufficient volume of staining solution to completely submerge the gels. pour the 100x stain into a storage bottle and save it for future use. Rinse gels Transfer the gels into a large container containing 500–700 ml of clean. special attention must be given when handling it. We highly recommend using a large spatula or other supportive surface to transfer the gel from one container to another. Each student team may utilize separate washing containers for each wash step.Staining DNA with Fast Blast DNA Stain (Laboratory Procedure) There are two protocols for using Fast Blast DNA stain in the classroom. at each student workstation. Using a funnel. The stain can be reused at least 7 times. This is easily accomplished by holding the base of the gel tray in one hand and gently pushing out the gel with the thumb of the other hand. We recommend using 120 ml of 100x Fast Blast to stain two 7 x 7 cm or 7 x 10 cm agarose gels in individual staining trays provided in Bio-Rad's education kits. 10 seconds 36 Student Manual . 1. Fast Blast DNA stain (500x) should be diluted to a 100x concentration. STUDENT MANUAL LESSON 2 2–3 minutes 3.
This is due to Fast Blast stain molecules migrating into the gel and binding more tightly to the DNA. additional washes in warm water may be necessary. The result will be a flat. the gel will dry completely at room temperature after 2–3 days. 5 minutes 6. Cut your gel from top to bottom to remove the lanes that you did not load samples into. Gently rock or shake the gel on a rocking platform for 5 minutes. transparent. Place your gel on a light background and record your results by making a diagram as follows. Wash gels Perform a second wash as in step 4. As the gel dries it will bond to the film but will not shrink. you may transfer the gel to 1x Fast Blast stain for overnight staining. If no rocking platform is available. warm tap water. i. trace the wells and band patterns onto the plastic sheet to make a replica picture of your gel. a. The bands may appear fuzzy immediately after the second wash. Wash gels Transfer the gel into a large container with 500–700 ml of clean. gels can be photocopied on a yellow piece of transparent film for optimal contrast. Record results Pour off the water and examine the stained gels for expected DNA bands. (Water will form beads on the hydrophobic side of a piece of gel support film.) Center the gel on the film and remove bubbles that may form between the gel and film. 5 minutes 5. but will begin to develop into sharper bands within 5–15 minutes after the second wash. Place the film on a paper towel and let the gel dry in a well-ventilated area. Trim away any unloaded lanes with a knife or razor blade. and durable record for the experiment. ii. Remove the plastic sheet for later analysis. move the gels gently in the water once every minute. Gel support film Student Manual 37 STUDENT MANUAL LESSON 2 . See Protocol 2. If you cannot complete the destaining in the allocated time. but do not wash the gel in water overnight. Place a clear sheet of plastic sheet or acetate over the gel. Dry the agarose gel as a permanent record of the experiment. With a permanent marker. leaving only lanes 1–4. Place the gel directly upon the hydrophilic size of a piece of gel support film. If left undisturbed on the support film. Alternatively. b. To obtain maximum contrast.4. Destain to the desired level. making sure to avoid direct exposure to light.
Stain gels (overnight)* Pour 1x stain into a gel staining tray.Protocol 2: Overnight Staining of Agarose Gels in 1x Fast Blast DNA Stain For overnight staining. but at least 8 hours of staining is recommended for complete visibility of stained bands. This is easily accomplished by holding the base of the gel tray in one hand and gently pushing out the gel with the thumb of the other hand. If no rocking platform is available. 2. add a sufficient volume of staining solution to completely submerge the gels. Following DNA electrophoresis. agarose gels must be removed from their gel trays before being placed in the staining solution. Label the staining tray with your initials and class period. add more 1x staining solution to completely submerge the gels. Because the gel is fragile. . agitate the gels staining tray a few times during the staining period. Fast Blast DNA stain (500x) should be diluted to a 1x concentration. You will stain 2 gels per tray. You should begin to see DNA bands after 2 hours. STUDENT MANUAL LESSON 2 38 Student Manual Stain overnight * It is crucial that you shake gels gently and intermittently while performing the overnight staining in 1x Fast Blast stain since smaller fragments tend to diffuse without shaking. We recommend using 120 ml of 1x Fast Blast to stain two 7 x 7 cm or 7 x 10 cm agarose gels in individual staining trays provided in Bio-Rad's education kits. Place the staining tray on a rocking platform and agitate overnight. If alternative staining trays are used. special attention must be given when handling it. If necessary. 1. Remove the gel from the gel tray and carefully slide it into the staining tray containing the stain.
making sure to avoid direct exposure to light. gels can be photocopied on a yellow piece of transparent film for optimal contrast. the gel will dry completely at room temperature after 2–3 days. Place your gel on a light background and record your results by making a diagram as follows. a. Alternatively. The gels can be analyzed immediately after staining. trace the wells and band patterns onto the plastic sheet to make a replica picture of your gel. b. Cut your gel from top to bottom to remove the lanes that you did not load samples into. Remove the plastic sheet for later analysis. Place the gel directly upon the hydrophilic size of a piece of gel support film. As the gel dries it will bond to the film but will not shrink. ii. With a permanent marker. If left undisturbed on the support film. However. (Water will form beads on the hydrophobic side of a piece of gel support film. transparent. Dry the agarose gel as a permanent record of the experiment. i. Trim away any unloaded lanes with a knife or razor blade. Student Manual 39 STUDENT MANUAL LESSON 2 . The result will be a flat. Gel support film Note: Avoid extended exposure of dried gels to direct light to prevent band fading.) Center the gel on the film on a paper towel and let the gel dry in a well-ventilated area. Place a clear sheet of plastic sheet or acetate over the gel.3. DNA bands will reappear if the dried gels are stored in the dark for 2–3 weeks after fading. Record results No destaining is required after staining with 1x Fast Blast. leaving only lanes 1–4. and durable record for the experiment.
Assuming a circular piece of DNA (plasmid) was used as starting material. If this were a fingerprinting gel. Based on your analysis of the example gel drawing on page 35. which DNA samples appear to have been “cut” into the same number and size of fragments? STUDENT MANUAL POST-LAB ACTIVITY 9. 6. What would be a logical explanation as to why there is more than one band of DNA for each of the samples? 4. What caused the DNA to become fragmented? 5. What can you assume is contained within each band? 2. how many samples of DNA can you assume were placed in each separate well? 3.Post-Lab: Thought Questions 1. which ones? Describe the evidence that supports your conclusion. how many restriction sites were there in lane three? 8. what is your conclusion about the DNA samples in the drawing? Do any of the samples seem to be from the same source? If so. Which sample has the smallest DNA fragment? 7. Which of the DNA samples have the same number of restriction sites for the restriction endonucleases used? Write the lane numbers. From the gel drawing on page 35. 40 Student Manual .
Dried electrophoresis gel Student Manual 41 STUDENT MANUAL POST-LAB ACTIVITY . Tracing of electrophoresis gel l f l h G l Attach the dried gel showing the banding patterns from the DNA electrophoresis below. Attach the plastic sheet tracing of the banding patterns from the DNA electrophoresis below.Post-Lab: Analysis of Results If the overnight staining protocol was used to stain gels. record your results and dry gels as described in the gel staining procedures in Lesson 2 page 38.
and then follow the graph line to over to the y-axis. Do this for all crime scene and suspect fragments. plot distance versus size for bands 2–6. 2. Is there a suspect that matches the crime scene? How sure are you that this is a match? 42 Student Manual . Measure the distance from the bottom of the well to the center of each DNA band and record your numbers in the table on the next page. read up to the standard line. Using a ruler. a standard curve is created using the distance (x-axis) and fragment size (y-axis) data from the known HindIII lambda digest (DNA marker). Where the graph line meets the y-axis. To estimate the size of an unknown crime scene or suspect fragment. 4. STUDENT MANUAL POST-LAB ACTIVITY 5. draw a line of best fit through the points. measure the distance (in mm) that each of your DNA fragments or bands traveled from the well. You might want to draw a light pencil mark from the x-axis up to the standard curve and over to the y-axis showing what you’ve done. Which graph provides the straightest line that you could use to estimate the crime scene or the suspects’ fragment sizes? Why do you think one graph is straighter than the other? 3. or would you want some more accurate measurement? In order to make the most accurate comparison between the crime scene DNA and the suspect DNA. should be used to estimate the DNA fragment sizes of the crime scene and suspects. Locate that distance on the x-axis of your standard graph. would you want to rely on a technician’s eyeball estimate of a match. other than just a visual match. On each graph. find the distance that fragment traveled. The data in the table will be used to construct a standard curve and to estimate the sizes of the crime scene and suspect restriction fragments. Compare the fragment sizes of the suspects and the crime scene. Decide which graph. Extend the line all the way to the righthand edge of the graph.Quantitative Analysis of DNA Fragment Sizes If you were on trial or were trying to identify an endangered species. To make an accurate estimate of the fragment sizes for either the crime scene or suspect DNA samples. Justify your selection. this is the approximate size of your unknown DNA fragment. linear or semilog. From that position on the x-axis. Using both linear and semilog graph paper. This is described below: 1. a quantitative measurement of the fragment sizes needs to be completed.
557 4 4. size (bp) Distance (mm) Approx. size (bp) Distance (mm) Approx. size (bp) Distance (mm) Approx.027 43 STUDENT MANUAL POST-LAB ACTIVITY . size (bp) 1 23. size (bp) Distance (mm) Approx.322 6 2.361 5 2.416 Student Manual Electrophoresis data: Measure the distance (in millimeters) that each fragment traveled from the well and record it in the table. Remember: some lanes will have fewer than 6 fragments.Lambda/HindIII size marker Band Distance (mm) Actual size (bp) Distance (mm) Approx. in base pairs. by comparing its position to the HindIII lambda DNA markers. Estimate its size.130 2 9. size (bp) Distance (mm) Crime Scene Suspect 1 Suspect 2 Suspect 3 Suspect 4 Suspect 5 Approx. 3 6.
000 10.000 Size. base pairs 1. mm STUDENT MANUAL POST-LAB ACTIVITY 44 Student Manual .Semilog Graph Paper 100.000 100 0 5 10 15 20 25 30 Distance.
000 5.000 0 0 5 10 15 20 25 30 Distance. base pairs 15.000 Size. mm Student Manual 45 STUDENT MANUAL POST-LAB ACTIVITY .Graph Paper 25.000 20.000 10.
What can you assume is identical about the molecules at that location? 6. What are we trying to determine? Restate the central question.Post Lab: Interpretation of Results 1. What caused the DNA to become fragmented? 4. Which of your DNA samples were fragmented? What would your gel look like if the DNA were not fragmented? 3. Based on the above analysis. What determines where a restriction endonuclease will “cut” a DNA molecule? 5. STUDENT MANUAL POST-LAB ACTIVITY 46 Student Manual . do any of the suspect samples of DNA seem to be from the same individual as the DNA from the crime scene? Describe the scientific evidence that supports your conclusion. 2. Do any of your suspect samples appear to have EcoRI or PstI recognition sites at the same location as the DNA from the crime scene? 7. A restriction endonuclease “cuts” two DNA molecules at the same location.
they can be joined (or ligated) to a piece of DNA from any organism (foreign DNA) that has been cut with the same enzyme. Plasmid mapping has revolutionized molecular biology and paved the way for the biotechnology industry. Purified restriction enzymes can be used in the laboratory to cut DNA isolated from any organism not just phage DNA. How do the cuts from one restriction enzyme overlay with cuts from a second restriction enzyme? There are clues to see how to overlap them: Do any of the first fragments remain uncut Student Manual 47 STUDENT MANUAL EXTENSION ACTIVITY 1 . this activity utilizes plasmid DNA to simulate how real DNA fingerprints are analyzed. the number of fragments represents the number of cuts or restriction sites. After plasmids are cut with a restriction enzyme. Plasmids often carry genes encoding resistance to antibiotics which gives bacteria a selective advantage over their competitors. Every hybrid plasmid now contains a copy of the piece of foreign DNA joined to it. were used together in a double digest. the restriction enzyme. or restriction fragment length polymorphisms (RFLP). Plasmids are circular. How many fragments are there now? The most informative part of plasmid mapping comes from using logic to overlap the information from two single digests with information obtained from a double digest. as the basis for much of biotechnology. The background material can be used to construct a plasmid map Since the plasmids are circular. Although real-world DNA fingerprinting is performed on genomic DNA. Scientists routinely take advantage of plasmid DNA and a natural bacterial defense mechanism. In the forensic DNA fingerprinting lab two restriction enzymes. The crime scene and suspect DNA samples in this kit were created by inserting lambda phage DNA that had been digested with the PstI restriction enzyme into PstI-digested plasmid pTZ18U. Restriction enzymes allow bacteria to destroy DNA from invading bacteriophages (phages) which are viruses that infect and destroy bacteria. The resulting fragments were run on a gel to solve the “who done it”. non-chromosomal pieces of DNA that can replicate in bacteria. We say that the foreign piece of DNA has been “cloned” and the plasmid DNA that carries it is called a “vector”. This technique allows molecular biologists to quickly evaluate the success of cloning experiments as well as to easily identify plasmids and associated traits in different organisms. striking banding patterns. Plasmids can be mapped or described in terms of location of restriction sites using simple experiments and logic.Extension Activity 1: Plasmid Mapping Plasmids and Restriction Enzymes This lesson will demonstrate the principles of plasmid mapping by examining restriction digestion patterns of plasmids used in the laboratory section of the kit and determining the position of restriction enzyme recognition sites in the plasmids by use of logic. except that the foreign DNA that was incorporated is also perpetuated. How many fragments are there? Cut the same rubber band again. Restriction maps of some of the crime scene and suspect plasmids are included on page 48. Recombinant plasmids were selected that gave distinct. Restriction enzymes recognize specific DNA sequences within the phage DNA and then cut the DNA at that site. A hybrid plasmid can replicate itself in bacteria similar to the original plasmid. take a rubber band and cut it once. Fragmented DNA no longer poses a threat to bacterial survival. The general procedure is to cut (digest) a plasmid with two restriction enzymes separately (two single digests) and the together (a double digest). Sizes of the resulting DNA fragments are determined then one uses logic to determine the relative location of the restriction sites. The resulting hybrid DNA plasmid can be put into (transformed) bacterial cells. PstI and EcoRI. To visualize this. when digested with restriction enzymes PstI and EcoRI and analyzed on an agarose gel.
In addition it shows the Origin of Replication (Ori). there is an arbitrary zero point. cut (linear) or uncut and twisted (supercoiled). Plasmids Used In This Lesson Plasmid maps show the positions (numbered by DNA base pairs) of sites where the plasmid may be cut by particular restriction enzymes.STUDENT MANUAL EXTENSION ACTIVITY 1 with the second enzyme? Do the sizes of any of fragments from the double digest add up the size of a fragment from a single digest? Do any of the fragments seem to remain the same size after being cut by the second enzyme? A simple example is shown below: 1000 bp plasmid example DNA size marker 1000 bp 700 bp 500 bp 300 bp 200 bp Digested with Enzyme 1 Digested with Enzyme 2 1000 bp 700 bp 300 bp 500 bp 300 bp 200 bp Digested with Enzyme 1 & 2 Undigested 1000bp Note that the two 1000 bp fragments (undigested sample and sample digested with enzyme 2) might not run at exactly the same distance on an agarose gel. but other enzymes could also have been used to cut these plasmids. Refer to the plasmid maps on page 49 for more detail. the origin of replication site. the genes present. All five of the plasmids used in the DNA fingerprinting activity were constructed from the same pTZ18U plasmid parent but had different foreign fragments of DNA inserted into them. The restriction sites are marked on the map with a number that indicates the location of the site. Since the plasmid is circular. Reading a Plasmid Map A plasmid map includes information on the size of the plasmid. Fragment sizes can then be calculated by simple subtraction (and in some cases addition) between points on the plasmid. In addition. may not be detected by the DNA stain or may run off the end of the gel. The name of the plasmid and its size of DNA in bp is shown inside the circle. In the DNA fingerprinting exercise. the gene encoding beta lactamase (the enzyme that gives the bacteria resistance to the antibiotic ampicillin) and the location where foreign DNA from the lambda bacteriophage was inserted. and restriction sites for restriction enzymes. only two restriction enzymes were used. fragments that are very small. All of the restriction sites are indicated with a number between zero and the total base pairs in the plasmid. 48 Student Manual . There is a small difference in migration if a fragment of the same size is uncut (circular). Also fragments that are very similar in size may migrate together in an agarose gel and it may not be possible to distinguish between them.
Sample Plasmid Maps Plasmid S2 Plasmid S5 Student Manual 49 STUDENT MANUAL EXTENSION ACTIVITY 1 .
How many sites are there? What is their location? If PvuII was used to cut (digest) this plasmid.STUDENT MANUAL EXTENSION ACTIVITY 1 Reading a Plasmid Map Questions 1. Which plasmid. DNA fragment size is calculated by subtracting the site locations from each other. it is not just a simple subtraction!). find the restriction sites for the enzyme PvuII. how many fragments would it make? 4. Student Manual 50 . Using plasmid S2 as an example. is the biggest and what is its size? 3. If the fragments from the plasmid S2 digested with PvuII were run on an agarose gel. (Note: if a fragment contains the 0 point of the plasmid. 2. S2 or S5. what would they look like? Draw the gel and label the fragments and their sizes. Next determine the size of the fragments created when plasmid S2 is cut by PvuII. From the map of plasmid S2 list all the restriction enzymes that would cut this plasmid. How big are the fragments from plasmid S2 that is cut with PvuII? The fragment sizes should add up to the total for that plasmid (5869 bp). 5.
EcoRI and PstI. Also given are the data for single digests by the individual enzymes. Now you can determine the fragment sizes of the plasmids when cut with the two enzymes. what would the gel look like? Draw a gel and the fragment sizes if digested by EcoRI alone. PstI alone and by EcoRI and PstI together. How does your diagram in question 7 compare to what was observed in your gel after the experiment? Indicate a reason for why your data in question 7 might be different from the actual experimental data seen from lesson 2. The charts below show the sizes of fragments that will be generated when plasmid S5 has been digested with the indicated restriction enzyme. First examine the results for plasmid S5 as an example. Enzymes Fragments Plasmid S2 (5869 bp) EcoRI PstI Both Total 7. If plasmid S2 was digested and run on an agarose gel. EcoRI alone or by both PstI and EcoRI. Student Manual 51 STUDENT MANUAL EXTENSION ACTIVITY 1 .6. The data given in the following table are for the double digest using both EcoI and PstI. Indicate the sizes of the fragments that would be generated if the plasmid were a digest by PstI alone. 8. The numbers in the columns under each enzyme represent the sizes of all of the fragments that are formed when each enzyme is used for a single digest. Mapping the Plasmid The first step in mapping a plasmid is to determine how many times a restriction site is found on that plasmid.
How big is plasmid S5? Add the fragments in each column. The total should add up to the size of the plasmid. Why? 2.STUDENT MANUAL EXTENSION ACTIVITY 1 Plasmid S5 Enzymes Fragments Plasmid S5 (9481 bp) EcoRI PstI 9481 2860 2838 1986 1093 468 164 72 Both 2838 2817 1986 1093 468 164 72 43 Total Mapping the Plasmid Questions 1. How many fragments are there? Did the enzyme cut the plasmid. Look at the data from the EcoRI digest of plasmid S5. or did it remain as a circle? How could you tell? Student Manual 52 .
Shown above is the data generated from digestion of plasmid S3 with EcoRI and PstI. Compare the data from the PstI digest of plasmid S5 with that of the EcoRI digest. Which fragment of PstI digested plasmid S5 was shortened by a cut with EcoRI? How do you know this? 6. It is easier to map plasmid S3. Plasmid S3 (7367 bp) EcoRI PstI 6504 4507 863 2860 Enzymes Fragments Both 3687 2817 820 43 Total 7. How many fragments are there when EcoRI and PstI are used to digest plasmid S5? Does that answer the question of whether or not EcoRI cut the plasmid? Why? 5. it would be very difficult to place all the restriction sites in order. How many fragments are there? How many restriction sites are there for PstI? 4.3. With the data. Draw the PstI fragment that is cut with EcoRI in plasmid S5 to demonstrate how the fragment was cut with EcoRI. Plasmid S5 is difficult to completely map because of the numerous PstI restriction sites. How many times did EcoRI cut plasmid S3? What are the fragment sizes? Student Manual 53 STUDENT MANUAL EXTENSION ACTIVITY 1 . Restriction mapping is an exercise in critical thinking and logic.
Is there another possible order of restriction sites on plasmid S3 digested with both PstI and EcoRI? How might you resolve these possibilities? 12.STUDENT MANUAL EXTENSION ACTIVITY 1 8. The data from the EcoRI digest of plasmid S3 indicate that the fragments are not equal. Draw a possible map and label the EcoRI sites and the sizes of the fragments. When the gels were run for this experiment. 11. Now draw an approximate map of the PstI sites on plasmid S3 and label the PstI sites and the sizes of the fragments. Mark sizes of each fragment and name the restriction sites on your figure. 9. 10. Draw a circular map of plasmid S3 digested with both PstI and EcoRI. Which band is missing from your gel? Why? Student Manual 54 . there were only three bands for plasmid S3.
and once transformed can produce a human protein product. plasmids with inserted foreign DNA may be put into a bacteria (transforming them). A self-replicating plasmid contains its own origin of replication (Ori). The parent plasmid pTZ18U used for plasmid construction is 2860 bp in size. Five plasmids were constructed from a parent plasmid. but others replicate independently producing hundreds of copies of the plasmid within one cell. The biotech industry is in part based on this principle. plasmid S1 contains lambda sequence 20285–22425. As you look at the different plasmids. For example. This exercise is based on plasmids used in the forensic DNA fingerprinting lab. Lambda phage DNA was also digested with the PstI restriction enzyme and the resulting fragments were then inserted into the PstI – digested pTZ18U plasmid. The five plasmids constructed for the DNA fingerprinting lab were all based on this plasmid. causing the bacteria to transcribe and translate that message into a protein product. It has numerous restriction sites (see diagram on pages 56–57). Bacteria can be given human genes via a plasmid. Plasmids are made of DNA just like the bacterial genome. and then were cut (digested) to make fragments of different sizes for a gel analysis.Extension Activity 2: Constructing a Plasmid Some plasmids replicate when the bacterial genomic DNA replicates. notice that each plasmid contains different fragments of lambda phage DNA. Student Manual 55 STUDENT MANUAL EXTENSION ACTIVITY 2 . The pTZ18U plasmid was digested with the PstI restriction enzyme. How were the plasmids constructed? Could one have made the plasmids differently? Can one predict fragment sizes using plasmid maps constructed from the lambda bacteriophage genome? In this activity you will design a new plasmid. Since DNA is universal in all living things. Many plasmids also contain genes that give antibiotic resistance to bacteria.
STUDENT MANUAL EXTENSION ACTIVITY 2 Student Manual Plasmid S1 Plasmid S4 56 .
Parent Plasmid pTZ18U 57 Student Manual STUDENT MANUAL EXTENSION ACTIVITY 2 .
Constructing a Plasmid Questions 1. Where is the EcoRI site on the parent pTZ18U plasmid? 6. Which segment will you use? Student Manual 58 .000 base pairs in size. Look at plasmid S4.000 base pairs but not more than 10. Remember that the parent plasmid is 2860 bp in size. Where is the PstI site on the pTZ18U plasmid? 2. 4. What segment of lambda was added to that plasmid? Were any PstI restriction sites added to the plasmid with the inserted fragment of lambda DNA? 5. After looking at the plasmid map and also the lambda phage map. Choose a segment of lambda bacteriophage genome that could be cut out by the EcoRI enzyme. You will use EcoRI for the construction and need to refer to the lambda bacteriophage genome map that includes the EcoRI sites. Look at plasmid S1. can you determine how many PstI restriction sites were added to the plasmid because of the inserted lambda phage DNA fragment? Note that it is possible for these extra PstI sites to have been added if the original restriction digestion was done for a short time so that not all PstI sites would have been completely cut in every piece of lambda phage DNA. Now let us create a different plasmid from the parent plasmid. What segment of the lambda bacteriophage has been inserted? STUDENT MANUAL EXTENSION ACTIVITY 2 3. You must make a plasmid that is at least 5.
10. How big is your new plasmid? Give the positions of the restriction sites in your new plasmid a number indicating the location. PstI alone iii. EcoRI alone ii. EcoRI and PstI together (a double digest) 9. Draw your new plasmid with the insert of your choice. Remember that the first EcoRI site will still be position 255 as it is in the parent pTZ18U plasmid map. 8. Be sure to include the restriction sites for PstI and EcoRI in your drawing. Draw an agarose gel for each of these digests and label the fragment sizes.7. How many restriction sites are there now for PstI in your new plasmid? Predict what fragments you would generate if you were to digest your plasmid with: i. The lambda phage fragment can be inserted into the host plasmid in either orientation – forwards or backwards. How could you use plasmid mapping to determine in which orientation your fragment was inserted? Use a diagram in your explanation. 59 Student Manual STUDENT MANUAL EXTENSION ACTIVITY 2 .
however. and conservation biology not only to determine the identity of individuals but also to determine relatedness. scientists are learning a great deal about our evolutionary history from DNA analysis. 3. a process that uses DNA to show relatedness or identity of individual humans. These are.Appendix A Alternative DNA Fingerprinting Scenarios DNA typing. In addition. and other features with photographs. Using portable testing equipment. This process has been used to free innocent suspects. identify stolen animals. It is also being used to find genetic linkages to inherited diseases. Food identification (endangered species identification). Each of the following paragraphs describes a scenario in which DNA has been used to show how individuals are related to each other. or animals. Have your students research a scenario that is interesting to them and present their findings to the class. 2. Czar Nicholas II and his family were executed by the Bolsheviks in 1918. Hamburger has been shown to often be a mixture of pork and other non-beef meats. or to show that a person is (or is not) the perpetrator of a crime. These scenarios provide a context for using DNA typing for use in teaching molecular biology. Statistics show that about 1/3 of all sexual assault suspects are freed as a result of DNA testing. plants. The purity (or impurity) of ground beef has been proven using DNA typing. many times. conservation biology. The applications of DNA typing. DNA from the bones was compared to that of known descendants and it was determined that the bones do belong to the czar and his family (“Identification of the remains of the Romanov family by DNA analysis” Nature Genetics vol 6. are much broader than forensic science alone and are having a profound impact on our society. 1. Simpson case. was used to show that he could not have been the rapist. unavailable when he was convicted. and prove that whale meat has been substituted for fish in sushi. authorities have used DNA typing to determine that the fish served in sushi was really meat from whales and dolphins. 130. teeth. DNA typing is used in forensics. DNA typing has become the subject of much debate and interest because of its uses for forensics analysis in prominent criminal cases such as the O. reunite children with their relatives. Identifying human remains. Bones found in Russia are believed to be those of the Romanovs. anthropology. and DNA fingerprinting are all names for the same process. Scientists have used DNA typing to confirm that the body in the grave was (or was not) the person that was supposed to be there. Russia’s last imperial family.) APPENDIX A 60 . Accused and convicted felons set free because of DNA typing. endangered species that are protected by international law. A man imprisoned for 10 years was released when DNA testing. and biotechnology. It is used in times of war to help identify the remains of soldiers killed in combat. DNA profiling. 1994. J. Experts from around the world have been studying the bones to match skulls.
Proving paternity.4. her 18th birthday. he refused to admit being her father.) 6. 264:1775. . 1994). 25.” says Svante Paabo of the University of Munich.) 61 APPENDIX A Girls in Florida were discovered to have been switched at birth when one girl died of a hereditary disease.000 year old “Iceman” found in a melting glacier is most closely related to modern Europeans. (Morell. 9. born in the same hospital and about the same time she was born. 7. July 10. Many of the children born to the disappeared adults were kidnapped and adopted by military "parents" who claimed to be their biological parents.000 citizens of these countries who disappeared between 1975 and 1983. the child was placed in the home of its biological relatives. June 17." Science. abducted by special units of the ruling military and police. but as long as he lived. vol. vol. Eva Harris. DNA testing of families. "Pulling Hair from the Ground. vol. 266:1317–1318. the transferred children became integrated into their biological families with minimal trauma. 1943." Science. vol. After the man died. would be agonizing. DNA typing has shown that the 5. (Science. Studying relatedness among ancient peoples. Identifying birth parents (paternity testing). 5. Determining relatedness of humans. 7. A woman.) The DNA typing evidence also “removes all the suspicions that the body was a fraud—that it had been placed on the ice. ("A Child of Rape Wins Award from Estate of Her Father. ("Iceman Gets Real. 264:1669. Nov. 1994. Virginia. 265:741-745 August 1994." New York Times. "A Personal Technology Transfer Effort in DNA Diagnostics. Identifying organisms that cause disease. DNA found at archeological sites in western Montana is being used to help determine how many related groups of people (families) lived at a particular site. The disease was not in her family. has helped scientists in Nicaragua and Ecuador to learn to use DNA technology to detect tuberculosis. and identify the dengue virus and various strains of Leishmania. became pregnant. June 17. but was known to be in the family of another girl." Science. but who had reared the child for years. (Marcia Barinaga.) 8. DNA testing proved that she was his daughter and she was granted a half of his estate. DNA testing of families has been used in Argentina and El Salvador to identify the children of at least 9. It was feared that transferring a child from its military "parents" who were kidnappers. a UCSF scientist. In practice. 1994. Other available tests cause waits of many weeks while disease organisms are cultured and sent to foreign labs to be identified. raped by her employer on Jan. 1994. The child knew who her father was. After genetic testing of the extended family revealed the true identity of a child.
(DNA fingerprints. and many so-called sub-species may all be part of a single species. DNA testing of plant material puts murderer at the scene. New Scientist. a fingerprint from the patient’s blood shows the donor’s bands.000 immigration applications from the wives and children of Bangladeshi and Pakistani men residing in the United Kingdom. Confirming relatedness among animals. Britain’s Home Office received 12. DNA fingerprinting has been used as proof of paternity for immigration purposes. The accused had admitted he had given the victim a ride. Nov. If the transplant has worked." ("Our Ultimate Identity Card in Sickness and in Health. so the leukemia sufferer will die without a transplant of healthy marrow.10. J. Scientists who extracted DNA from the hair of chimpanzees throughout Africa now have evidence that there might be a third species of chimpanzee. Genetic testing showed that DNA in the seed pod exactly matched the DNA of a plant found at the scene of the murder. Two small seed pods caught in the bed of his pick-up truck put an accused murderer at the murder scene.) 11. At the same time they have learned things about chimp behavior and kinship patterns that would have once taken years to theorize. "DNA fingerprinting can help doctors to monitor bone marrow transplants." in "Inside Science". vol. But if the cancerous bone marrow has not been properly destroyed. 16. The male chimps’ relatedness may explain why. source unknown: Based on A. 1991. Blood tests can also be inconclusive. APPENDIX A 62 .) 12. 1985." Nature. Leukemia is a cancer of the bone marrow and the diseased marrow must be removed. The burden of proof is on the applicant. "Positive Identification of an Immigration Test-Case Using Human DNA Fingerprints. Proving relatedness of immigrants. They discovered a group of chimps living in western Africa to be genetically distinct from the chimps living in other parts of Africa. but DNA fingerprinting results are accepted as proof of paternity by the Home Office. suggesting that the group may be an endangered species. then the cancerous cells multiply rapidly and the patient’s own bands predominate. 13. Determining effectiveness of bone marrow transplants. 317:818–819.. Jeffreys et al. Doctors can quickly tell whether the transplant has succeeded by DNA typing of the patient and the donor. the males are quite friendly to each other. but establishing the family identity can be difficult because of sketchy documentary evidence. The have discovered that male chimps living in a given area are often as closely related as half-brothers. The bone marrow makes new blood cells. In 1986. unlike other primates. but he denied ever having been near the crime scene.
Appendix B Prelab Activity 1 A Review of Restriction Enzymes DNA consists of a series of nitrogenous base molecules held together by weak hydrogen bonds. Fig. (A.. Ts. Refer to the figures below to review the structure of a DNA molecule... These base pairs are in turn bonded to a sugar–phosphate backbone. the base-pair sequence might appear as: Read to the right----> A C T C C G T A G A A T T C. Describe any relationship you can see. guanine and cytosine.T G A G G C A T C T T A A G <----Read to the left Look at the linear sequence of bases (As. 3.) on each of the strands: 1. 1. and C: Remember the base-pairing rule is A-T and G-C). 2. Do you notice any grouping of bases that when read toward the right on the upper strand and read to the left on the bottom strand are exactly the same? 63 APPENDIX B . T. etc. The Structure of DNA If a segment of DNA is diagrammed without the sugars and phosphates. The four different nitrogenous bases are adenine. Now look at the upper sequence of bases and compare it to the lower. thymine.> <. Compare the bases in the upper portion of the molecule to those in the lower portion.. G. Describe any pattern you might see in the upper sequence of bases...
This could be considered a very primitive immune system. are fairly common along the DNA molecule. whereas other restriction enzymes make a cut across both strands at the same place creating double stranded DNA fragments with “blunt” ends. How many bases are still paired to the left of the “cut”? 5. These sequences. Restriction enzymes search the viral DNA for specific palindromic sequences of base pairs. These viruses inject their own DNA into bacteria and force the bacteria to multiply the DNA. at the DNA site where they cut. called a “sticky” end. CTTAAG (read toward the left). called palindromes. Look at the DNA sequence below: Palindrome G T A G A AT T C A T T C A C G C A C A T C T TA A G T A A G T G C G T ✄ GTAG CATCTTAA Fragment 1 ✄ Restriction site AATTCATTCACGCA GTAAGTGCGT “sticky” end cut Fragment 2 APPENDIX B A restriction enzyme cut the DNA between the G and the A in a GAATTC palindrome. The actual sequence of DNA is called a restriction site.You may have discovered that the sequence of base pairs is seemingly random and that the two strands are complementary to each other: As are paired with Ts and Cs are paired with Gs. Bacteria prevent destruction of their own DNA by modifying certain DNA bases within the specific enzyme recognition sequence. Counting the number of paired bases. has a counterpart in the lower strand. 4. Example sequences are: GAATTC CTTAAG AAGCTT TTCGAA CTGCAG GACGTC When such a sequence is looked at together with its complementary sequence the group reads the same in both directions. Restriction Enzymes — Molecular Scissors Viruses called bacteriophages frequently infect bacteria. which allows them to protect their own DNA while cutting up viral DNA. such as GAATTC. How many bases are still paired to the right of the “cut”? 6. How could you describe the size of each fragment in terms of the number of base pairs in the fragment? 64 . Bacteria have evolved restriction enzymes. and cut the DNA at these sites. Some restriction enzymes may leave a short length of unpaired nucleotide bases. You may have also noticed that a portion of the top strand GAATTC (read toward the right). to cut up and destroy the invading viral DNA. is the right fragment the same size as the left fragment? 7.
no matter what species the DNA comes from. and the restriction enzyme that recognizes that base sequence is present and digests the DNA. 8. then what can you say about the sizes of the fragments that will be produced when the DNA is digested with a restriction enzyme that recognizes that sequence? The table below shows palindromic sequences that are recognized by the enzymes that are used to digest the DNA you will be analyzing in this activity. how many DNA fragments will be produced? 9.An important feature of restriction enzymes is that each enzyme only recognizes a specific palindrome and cuts the DNA only at that specific sequence of bases. and the specific restriction enzyme will cut all those palindromes. Palindromic sequence GAATTC CTTAAG AAGCTT TTCGAA CTGCAG GACGTC Name of restriction enzyme that recognizes the palindrome EcoRI HindIII PstI 65 APPENDIX B . If the GAATTC palindrome repeats are randomly found along the DNA strand. A palindromic sequence can be repeated a number of times on a strand of DNA. If the GAATTC palindrome is repeated four times on the same piece of linear DNA.
for restriction enzyme A. GAATT C CT TAAG • • • • • Palindromes can be detected by restriction enzymes Restriction enzymes cut the palindromes at restriction sites Restriction enzymes recognize specific palindromes Cutting DNA at restriction sites will produce DNA fragments Fragment size can be described by the number of base pairs a fragment contains Applying What You Have Learned A linear DNA molecule is represented below. DNA has two strands. 3. The DNA is represented by one line. Number each fragment. If the DNA molecule has two restriction sites.. Which fragment is the smallest? APPENDIX B 66 . Which fragment is the largest? 4.e. how many fragments would be produced if the DNA is cut by that enzyme? 2.Below is the summary of what we have learned so far: • A sequence on one strand of DNA and its complementary sequence on the other strand can form a palindrome i. although in actuality. 1.
5. Explain why only two fragments would be produced. 7. 67 APPENDIX B . 8. Label each fragment. In this diagram A and B are different palindrome sequences on a DNA strand. Only the restriction enzyme that recognizes site B is present. Draw a DNA molecule that has five randomly spaced restriction sites for a specific palindrome. Rank them in order of size from largest to smallest. How many fragments would be produced if each site were cut by that specific restriction enzyme? 6.
how would the gel appear? 3. those with the greater number of base pairs. Where would the larger fragments. Using the information given above. the … 68 . A solution containing the four fragments is placed in a well in an agarose gel. If it were possible to weigh each of the fragments. which one would be the heaviest? Why? APPENDIX B 4. Complete this rule for the movement of DNA fragments through an agarose gel. A piece of DNA is cut into four fragments as shown in the diagram. Imagine the gel as a strainer with tiny pores that allow small particles to move through it very quickly. toward the top of the gel or the bottom? Why? 2. A solution containing a mixture of DNA fragments of variable sizes is placed into a small well formed in an agarose gel that has a texture similar to gelatin. Have your teacher check your diagram before you proceed. An electric current causes the negatively-charged DNA molecules to move towards the positive electrode. Fragments that are the same size will tend to move together through the gel and form bands. The larger the DNA fragment. Scientists have used this fact to design a method that can be used to separate pieces of DNA. the DNA fragments will sort themselves out by size. be located. DNA is an acid and has many negative electrical charges. the slower they are strained through the gel.Prelab Activity 2 A Review of Electrophoresis Agarose gel electrophoresis is a procedure used to separate DNA fragments based on their sizes. however. The larger the size of the particles. Label each fragment with its corresponding letter. 1. After a period of exposure to the electrical current. draw (to the right) how you think the fragments might be separated. Suppose you had 500 pieces of each of the four fragments.
361 5.416 6. Label each fragment with its correct number of base pairs.This diagram represents a piece of DNA cut with HindIII at each of the restriction sites pointed to by the arrows. 23. Well Negative Positive 7. How many fragments were produced by the restriction enzyme HindIII? 6.027 2.130 2.322 9. The numbers represent the number of base pairs in each fragment. The two fragments with no length indicated will be too small to be visualized on the gel. On the gel diagram. 69 APPENDIX B .557 4. show how you believe these fragments will sort out during electrophoresis.
All samples contain the same bases: adenine. do all three samples contain the same bases? Describe your observations. What will you need to compare between these DNA samples to determine if they are identical or non-identical? The sequence of base pairs in each individual sample. Compare the “backbone” of sugar-phosphate arrangement in the side chains of all three figures. guanine and cytosine. thymine. 5.APPENDIX C Appendix C Instructor’s Answer Guide Pre-Lab Focus Questions: Introduction to DNA Fingerprinting 1. Are there any differences? The arrangement is identical for all three samples. what conclusions can you make about the similarities and differences of the DNA samples? The sugar phosphate arrangement is the same for all samples and so are the kind of bases. 4. In your attempt to analyze DNA samples from three different individuals. what is different is the arrangement of bases among the three samples. In the above figure. Are the bases paired in an identical manner in all three samples? Describe the pattern of the base pair bonding. 2. 3. 70 . The adenine is always bonded with thymine and the cytosine is always bonded with the guanine.
also some bases are unpaired. Write the base sequence of the DNA fragments on both the left and right side of the “cut”. 11 b) What is the length of the longer fragment ? 5. What differences are there in the two pieces? Each fragment is a different size. Indicate the size of the fragments [mention any discrepancy you may detect]. How many pieces of DNA would result from this cut ? 2 2. Consider the two samples of DNA shown below [single strands are shown for simplicity]: Sample #1: CAGTGATCTCGAATTCGCTAGTAACGTT Sample #2: TCATGAATTCCTGGAATCAGCAAATGCA If both samples are treated with a restriction enzyme [recognition sequence GAATTC] then indicate the number of fragments and the size of each fragment from each sample of DNA. One fragment is short and one is long. 4. a) The smaller fragment is 3 base pairs (bp). ATG TACTTAA A A T T C T C AA T T A C C T GAGTTAATGGA 3.Lesson 1 Restriction Digests of DNA Samples 1. Sample # 1 Sample # 2 # of fragments: 2 # of fragments: 2 List fragment size in ascending order: largest ——> smallest Sample # 1 17 bp fragment 11 bp fragment Sample # 2 23 bp fragment 5 bp fragment 71 APPENDIX C . DNA fragment size can be expressed as the number of base pairs in the fragment.
3. The DNA samples are clear. 2. 72 . colorless liquid samples. colorless liquids. Is there any observable difference between the samples of DNA? No.APPENDIX C Lesson 1 Restriction Digestion of DNA Samples Observation Questions 1. Describe the samples of DNA (physical properties). Describe the appearance of the restriction endonuclease mix. All samples appear similar. The restriction enzymes appear to be clear.
Yes. No. DNA + EcoRI/PstI enzyme mix: No visible change apparent in the tubes. No. Before you incubated your samples. Enzymes may have cut the DNA. After a 24 hour incubation period. Can you see any evidence to indicate that your samples of DNA were fragmented or altered in any way by the addition of EcoRI/PstI? Explain.Lesson 1 Restriction Digestion of DNA Samples Review Questions 1. 73 APPENDIX C . The reactions are at the molecular level and too small to be seen. They may be chemically changed but the changes may not be visible. No visible change apparent in the tubes but the enzymes may have cut the DNA. No visible change apparent in the tubes. is it still possible that the DNA samples were fragmented? Explain your reasoning. 4. In the absence of visible evidence of change. 3. 2. describe any visible signs of change in the contents of the tubes containing the DNA combined with the restriction enzymes. are there any visible clues that the restriction enzymes may have in some way changed the DNA in any of the tubes? Explain your reasoning.
DNA molecules are negatively charged. The electrophoresis apparatus creates an electrical field [positive and negative ends of the gel]. Which fragments are expected to travel the shortest distance [remain closest to the well]? Explain. To which pole of the electrophoresis field would you expect DNA to migrate (+ or -)? Explain. There is more resistance to their movement through the gel matrix. 74 . Smaller. There is less resistance to their movement through the gel matrix. Positive. What color represents the negative pole? The negative pole (or cathode) is black.APPENDIX C Lesson 2 Agarose Gel Electrophoresis Review Questions 1. they are "forced" to move through the gel matrix. After DNA samples are loaded in wells. Which size fragment (large vs small) would you expect to move toward the opposite end of the gel most quickly? Explain. 2. The positive pole (or anode) is red. 4. Larger. 3.
If so which ones? Describe the evidence that supports your conclusion. 7. then how many kinds (samples) of DNA can you assume were placed in each separate well? One. 8. 3. Two sites that cut the sample into two fragments. 2. Which sample has the smallest DNA fragment? The sample in lane 5 (S3).Post-Lab Thought Questions 1. and S2). how many restriction sites were there in lane three? Please note that the starting material was a circular piece of DNA. Which of the DNA samples have the same number of restriction sites for the restriction endonuclease used? Write the lane numbers. which DNA samples appear to have been “cut” into the same number and size of fragments? Lanes 2 and 4 (CS and S2). 9. From the gel drawing on page 35. 4. S1. Lanes 2. and 4 (CS. If this were a fingerprinting gel. 75 APPENDIX C . What would be a logical explanation as to why there is more than one band of DNA for each of the samples? The DNA must have been cut into fragments by restriction enzymes. 5. Based on your analysis of the example gel drawing on page 35. What probably caused the DNA to become fragmented? The chemical action of the restriction enzymes cutting at specific base sequences. what is your conclusion about the DNA samples in the photograph? Do any of the samples seem to be from the same source. 6. What can you assume is contained within each band? DNA fragments. 3. Assuming a circular piece of DNA (plasmid) was used as starting material. The DNA samples in lanes 2 and 4 (CS and S2) are from the same individual because they have identical restrictions sites that yield identical fragments.
Lambda/HindIII size marker Band Distance (mm) Actual size (bp) Distance (mm) Approx. size (bp) Distance (mm) Approx. size (bp) Distance (mm) Approx. size (bp)
Approx. size (bp)
Approx. size (bp)
Approx. size (bp)
*This fragment may appear faint if the markers were not heated to 65 ºC. Lamba HindIII digestion also generates bands of 564 and 125 bp that are usually too faint to see on a gel.
**The measured migration distance for these bands varies depending upon the thickness of the bands. The 2,817 bp bands in plasmids S4 and S5 are especially intense because they are actually two individual bands (2,817 bp and 2,838 bp) that are too close to be visibly separated. ***S4 and S5 DNA lanes may also contain a very faint band of 468 bp.
DNA Standard Band Migration
Size, base pairs
100 0 5 10 15 20
To estimate the size of any unknown crime scene or suspect fragment, you first need to determine the distances the specific fragment travelled. Locate the distance on the x-axis of your standard graph. For example, suspect 5, band 2 migrated 24 mm (A). From the 24 mm mark on the x-axis, read up to the standard line; when you intersect your standard curve, mark the spot with a shaded circle (B). Follow the intersect point over to the y-axis and determine where the graph line meets the y-axis this is the approximate size of the fragment (C). Therefore, suspect 5, band 2 is approximately 2,000 bp. Repeat this procedure for the crime scene and all suspects’ fragments. As you determine the approximate fragment sizes, fill in the data in the data table.
Size, base pairs
25,000 20,000 15,000 10,000 5,000 0 0 5
Fingerprinting Standard Curve: Linear
Post Lab Activity: Interpretation of Results 1. 3. Which of your DNA samples were fragmented? What would your gel look like if the DNA were not fragmented? The number of fragmented samples will vary. What can you assume is identical about the molecules at that location? The restriction sites are identical. Based on the above analysis. They will have one band on the gel if the DNA was not cut. They both produce similar banding patterns on the gel. Do any of your suspect samples appear to have EcoRI or PstI recognition sites at the same location as the DNA from the crime scene? The samples in lanes 2 and 5 match (CS and S3). 2. We are trying to determine if samples of DNA that we were provided with are from the same individual or from different individuals. 79 APPENDIX C . A restriction endonuclease “cuts” two DNA molecules at the same location. What determines where a restriction endonuclease will “cut” a DNA molecule? A special sequence of bases on the DNA called restriction sites. do any of the suspect samples of DNA seem to be from the same individual as the DNA from the crime scene? Describe the scientific evidence that supports your conclusion. 6. 5. The CS and S3 samples appear to be identical. What caused the DNA to become fragmented? The addition of restriction enzymes. What are we trying to determine? Restate the central question. 4. 7.
Indicate the sizes of the fragments that would be generated if the plasmid were a digest by PstI alone. ScaI 2. find the restriction sites for the enzyme PvuII. (Note: if a fragment contains the 0 point of the plasmid. how many fragments would it make? There are three sites for PvuII on plasmid S2. it is not just a simple subtraction!). 4. 1938 and 2514 bp. BamHI. How big are the fragments from plasmid S2 that is cut with PvuII? The fragment sizes should add up to the total for that plasmid (5869 base pairs). EcoRV. They are at position 55. 1993 and 3410. Now you can determine the fragment sizes of the plasmids when cut with the two enzymes. Using plasmid S2 as an example. EcoRI. EcoRI and PstI.APPENDIX C Extension Activity 1: Plasmid Mapping Reading a plasmid map 1. 2514 1938 1417 6. If the fragments from the plasmid S2 digested with PvuII were run on an agarose gel. The fragment sizes are 1417. 5. How many sites are there? What is their location? If PvuII was used to cut (digest) this plasmid. what would they look like? Draw the gel and label the fragments and their sizes. Which plasmid. EcoRI alone or by both PstI and EcoRI. From the map of plasmid S2 list all the restriction enzymes would cut this plasmid. Size is calculated by subtracting the site locations from each other. Next determine the size of the fragments created when plasmid S2 is cut by PvuII. PvuII. PstI. HindIII. is the biggest and what is its size? S5 is the largest (9481 bp) 3. Three fragments would be created if plasmid S2 was digested with PvuII. Plasmid S2 (5869 bp) EcoRI PstI 5869 2860 1700 1159 150 Enzymes Fragments Both 2817 1700 1159 150 43 Total 80 . S2 or S5.
The total should add up to the size of the plasmid.860 43 2. Which fragment of PstI digested plasmid S5 was shortened by an EcoRI cut? The 2860 bp fragment was cut into two fragments (2817 bp and 43 bp) 6. what would the gel look like? Draw a gel and the fragment sizes if digested by EcoRI alone. The fragments are cut from the plasmid and all the pieces together should be equal to the size of the original plasmid. Look at the data from the EcoRI digest of plasmid S5. Compare the data from the PstI digest of plasmid S5 with that of the EcoRI digest. .817 EcoRI 81 APPENDIX C 7. The additional fragment shows that EcoRI also cut the plasmid one time. PstI alone and by EcoRI and PstI together.EcoRI 5809 PstI 2860 1700 1159 150 43 Both Enzymes 2817 1700 1159 150 43 8. Draw the PstI fragment that is cut with EcoRI in plasmid S5 to demonstrate how the fragment was cut with EcoRI. How many fragments are there? How many restriction sites are there for PstI? There should be 7 fragments and therefore 7 restriction sites. They should be similar but the bands at 150 and 43 bp are too small to be observed on the gel. 3. Why? The plasmid is 9481 bp in size. How many fragments are there when EcoRI and PstI are used to digest plasmid S5? Does that answer the question of whether or not EcoRI cut the plasmid? Why? There should be 8 fragments when the plasmid is cut by both enzymes. If plasmid S2 was digested and run on an agarose gel. How big is plasmid S5? Add the fragments in each column. it is not possible to tell if the plasmid was cut although sometimes a circular (and possibly twisted) plasmid does not run through an agarose gel at the same place as a linear piece of DNA of the same size. Mapping the Plasmid Questions 1. or did it remain as a circle? How could you tell? There should be only one fragment. 5. How many fragments are there? Did the enzyme cut the plasmid. 2. 2. How does your diagram in question 7 compare to what was observed in your gel after the experiment? Indicate a reason for why your data in question 7 might be different from the actual experimental data seen from lesson 2. At first glance. 4.
APPENDIX C 7. The data from the EcoRI digest of plasmid S3 indicate that the fragments are not equal. With the data. Now draw an approximate map of the PstI sites on plasmid S3 and label the PstI sites and the sizes of the fragments. It is easier to map plasmid S3. Draw a possible map and label the EcoRI sites and the sizes of the fragments. Plasmid S5 is difficult to completely map because of the numerous PstI restriction sites. it would be very difficult to place all the restriction sites in order. 82 . 8. Draw a circular map of plasmid S3 digested with both PstI and EcoRI? Mark sizes of each fragment and name the restriction sites on your figure. 9. The fragments are 863 and 6505 bp in size. How many times did EcoRI cut plasmid S3? What are the fragment sizes? EcoR1 cut plasmid S3 twice. 10. Restriction mapping is an exercise in critical thinking and logic.
there were only three bands for plasmid S3. 83 APPENDIX C 11.Using the data from the double digest there are two possibilities (see diagram). When the gels were run for this experiment. Which band is missing from your gel? Why? The 43 bp band is so small that it does not bind enough Fastblast stain to be visible or it may have run off the gel. However the single digest with PstI would have fragments of 3687 and 3637 bp (not 4507 and 2860 bp as was seen). 12. Is there another possible order of restriction sites on plasmid S3 digested with both PstI and EcoRI? How might you resolve these possibilities? .
Choose a segment of lambda bacteriophage genome that could be cut out by the EcoRI enzyme.104–31. 5.804 bp insert 8664 bp total EcoRI 255. 5. can you determine how many PstI restriction sites were added to the plasmid because of the inserted lambda phage DNA fragment? Note that it is possible for these extra PstI sites to have been added if the original restriction digestion was done for a short time so that not all PstI sites would have been completely cut in every piece of lambda phage DNA.104. Where is the EcoRI site on the parent pTZ18U plasmid? The EcoRI site is at 255 bp.747.104 4. lambda 39. 5. 4.285–22.176 POSSIBILITY 2 Lambda 26.059 PstI 6.102 84 .898 PstI 1.454.878 bp insert 7.503 bp total EcoRI 255.617 was added. Two restriction sites for PstI were added: site 766 and site 3.604. Remember that the first EcoRI site will still be position 255 as it is in the parent pTZ18U plasmid map.226–26.738 bp total EcoEI 255. You must make a plasmid that is at least 5. Now let us create a different plasmid from the parent plasmid. 7. Which segment will you use? There are three possibilities: lambda 21.133 PstI 1.000 base pairs but not more than 10. How big is your new plasmid? Give the positions of the restriction sites in your new plasmid a number indicating the location. lambda 26. 6. Look at plasmid S4. Look at plasmid S1. All other options would make the plasmid larger than 10.226–26.168–44.747 5.972 5. After looking at the plasmid map and also the lambda phage map.APPENDIX C Extension Activity 2: Constructing a Plasmid Questions 1. You will use EcoRI for the construction and need to refer to the lambda bacteriophage genome map that includes the EcoRI sites. POSSIBILITY 1 Lambda 21. 5. 5. What segment of lambda was added to that plasmid? Were any PstI restriction sites added to the plasmid with the inserted fragment of lambda DNA? Lambda fragment 20. Where is the PstI site on the pTZ18U plasmid? Position 298 2. Remember that the parent plasmid is 2.104–31.643 bp insert 8.083.425 was added to plasmid S1.860 bp in size.000 base pairs in size. 3.943 POSSIBILITY 3 Lambda 39.168–44. No additional PstI sites were added. Be sure to include the restriction sites for PstI and EcoRI in your drawing. What segment of the lambda bacteriophage has been inserted? Lambda fragment 5. 5.218–9.972.000 bp. Draw your new plasmid with the insert of your choice.
2860 2817 85 APPENDIX C 8.226-26. POSSIBILITY 1 POSSIBILITY 2 POSSIBILITY 3 Lambda Lambda Lambda 21. Using possibility 1 from questions 8 and 9 above.104 26. POSSIBILITY 1 EcoRI PstI Both POSSIBILITY 2 EcoRI PstI Both POSSIBILITY 3 EcoRI PstI 8664 5804 Both 5804 5643 4878 3722 2860 3679 2817 1199 2860 4858 4815 2817 828 The band at 43 bp is too small to be seen.104–31. POSSIBILITY 1 POSSIBILITY 2 POSSIBILITY 3 Lambda Lambda Lambda 21.104-31. The double digests will show the same bands with either orientation but the PstI single digest will be different.226–26.972 3679 4815 5804 2817 2817 2817 1199 828 43 43 43 9.972 4016 3722 4858 3645 8664 iii. How many restriction sites are there now for PstI in your new plasmid? Predict what fragments you would generate if you were to digest your plasmid with: .168-44. PstI alone Two fragments for possibility 1 & 2 and one fragment with possibility 3.104 26.168-44.i. POSSIBILITY 1 POSSIBILITY 2 POSSIBILITY 3 Lambda Lambda Lambda 21.168–44. 10.747 39.104 26. In the second orientation it will give two bands at 6492 bp and 1242 bp.972 4878 2860 5643 2860 5804 2860 ii. The lambda phage fragment can be inserted into the host plasmid in either orientation – forwards or backwards. How could you use plasmid mapping to determine in which orientation your fragment was inserted? Use a diagram in your explanation.747 39. EcoRI alone Two fragments in each case since there are two EcoRI sites. Draw an agarose gel for each of these digests and label the fragment sizes. EcoRI and PstI together (a double digest) Four fragments for possibility 1 and 2 and three fragments with possibility 3.747 39.226-26. In the first orientation it will give two fragments of 4016 bp and 3722 bp.104-31.
86 . 7. Describe any pattern you might see in the upper sequence of bases. Describe any relationship you can see. how many DNA fragments will be produced? 5 9. 4. and the restriction enzyme that recognizes that base sequence is present and digests the DNA. G always pairs with C. Now look at the upper sequence of bases and compare it to the lower. How many base pairs are there to the left of the cut? 4 5. How could you describe the size of each fragment in terms of the number of base pairs in the fragment? Fragment 1 is a 4-base-pair fragment. is the right fragment the same size as the left fragment? No. Compare the bases in the upper DNA strand to those in the lower strand. Counting the number of base pairs. 2. There is no specific type of pattern associated with the upper sequence of bases. A always pairs with T. 3. then what can you say about the size of the fragments that will be produced when the DNA is digested with a restriction enzyme that recognizes that sequence? Random sized fragments will be produced. Do you notice any grouping of bases that when read toward the right on the upper strand and read to the left on the bottom strand are exactly the same? CTTAAG.APPENDIX C Prelab Activity 1 A Review of Restriction Enzymes (from Appendix B) 1. How many base pairs are there to the right of the cut? 10 6. If the GAATTC palindrome repeats are randomly spaced along the DNA strand. Fragment 2 is a 10-base-pair fragment. 8. it is larger. If the GAATTC palindrome is repeated four times on the same piece of linear DNA.
4. how many fragments would be produced. Draw a DNA molecule that has five randomly spaced restriction sites for a specific palindrome. 8. Which fragment would be the smallest? Fragment 2. The enzyme would cut only at site B. How many fragments would be produced if they were each cut by a restriction enzyme? 6 6. Label each fragment Answers will vary. Only the restriction enzyme that recognizes site B is present. 3. In this diagram A and B are different palindrome sequences on a DNA strand. 5. 87 APPENDIX C . Which fragment would be the largest? Fragment 3. Rank them in order of size from largest to smallest. 7.Applying What You Have Learned 1. for restriction enzyme A. producing two DNA fragments. Number each fragment. Answers will vary. Explain why only two fragments would be produced. if the DNA is cut by enzyme A? 3 2. If a DNA molecule has two restriction sites.
draw on the diagram how you think the fragments might be separated. toward the top of the gel or the bottom? Why? The large fragments would be towards the top of the gel because it is more difficult for the larger pieces to be strained through the gel. Suppose you had 500 pieces of each of the four fragments. the slower it migrates through an agarose gel. which one would be the heaviest? Why? Fragment D would be heaviest because it is the largest piece of DNA and would thus have the greatest mass. The larger the DNA fragment. how would the gel appear? There would still be only 4 bands present. 88 . 2. 4. If it were possible to weigh each of the fragments. A solution of the four fragments is placed in a well in an agarose gel. those with the greater number of base pairs. Label each fragment with its corresponding letter. Have your teacher check your diagram before you proceed. 1. 3.APPENDIX C Prelab Activity 2 A Review of Electrophoresis (from Appendix B) A piece of DNA is cut into four fragments as shown in the diagram. Complete this rule for the movement of DNA fragments through an agarose gel. Using the information given above. Where would the larger fragments. be located.
322 9.361 5. See gel above. Label each fragment with its correct number of base pairs.416 6. 6. however only 6 will be large enough to appear on the gel.416 6. The two fragments with no length indicated will be too small to be visualized on the gel. The numbers represent the number of base pairs in each fragment. show how you believe these fragments will sort out during electrophoresis.23. Well Negative 23. On the gel diagram.130 2. How many fragments were produced by the restriction enzyme HindIII? 8.557 4.322 2.557 4.027 Positive Agarose gel 7.361 2.027 2. . 89 APPENDIX C This diagram represents a piece of DNA cut with HindIII at each of the restriction sites pointed to by the arrows.130 9.
25x TAE. Monitor the movement of gel loading dye to get a relative idea of electrophoresis progress. The provided 50x TAE buffer is mixed with distilled water to yield the necessary concentrations for making agarose gels and electrophoresis running buffer.25x TAE to make agarose gels. add 625 ml of 1x TAE to 1. To run gels: Place the gel in an electrophoresis chamber and cover it with 0.25x TAE). For added convenience. To make 500 ml of 1x TAE from a 50x TAE concentrate. Note: Do not use 0.5 liter volume of 0.875 ml of distilled water. precast 1% agarose gels made with 1x TAE are available from Bio-Rad (catalog #161-3057EDU) Use 0.25x TAE buffer is required to run eight 7 x 10 agarose gels.Appendix D Fast Gel Protocol The DNA Fingerprinting gels can be run in under 20 minutes by using a reduced concentration of running buffer (0.25x TAE to make electrophoresis running buffer: A 2. Resolution is excellent and the gel can be run 33% faster.5 L of 0. doing so can lead to a loss of DNA resolution. Use 1x TAE to make agarose gels: Half a liter of 1x TAE is sufficient to make eight 7 x 10 cm agarose gels. the same concentration of buffer in the gel itself (1x TAE) and higher voltage (200 volts). add 10 ml of concentrated TAE to 490 ml of distilled water.25x TAE from a 1x TAE solution. APPENDIX D 90 . Run gels at 200 V for no more than 20 min. ensure the gel is submerged. To make 2.
Appendix E: Suspect Plasmid Maps Plasmid Maps Suspect 1 DNA Sample Suspect 2 DNA Sample 91 APPENDIX E .
Crime Scene/Suspect 3 DNA Sample APPENDIX E Suspect 4 DNA Sample 92 .
Suspect 5 DNA Sample 93 APPENDIX E .
APPENDIX E Plasmid Parent Vector 94 .
262: 5. 16 November 1991. Elizabeth Shakir. it is a technique used to amplify small amounts of DNA (in this case so that further analysis of the DNA can occur). Vol. pp 199–205.. 10. 7. March 31. 1994. DNA Profiling Fast Becoming Accepted Tool For Identification. Chemical and Engineering News. Oct. Biochemistry and Molecular Biology Education. Using Restriction Mapping to Teach Basic Skills in the Molecular Biology Lab."riff-lips" in biotech jargon. 5. 1990.. When Science Takes the Witness Stand. Is DNA Fingerprinting ready for the courts?. May 1990. William C. PCR means polymerase chain reaction. New Scientist. Thompson and Simon Ford.Pieces of DNA are cut with restriction enzymes into fragments of various lengths. 95 APPENDIX F . and Elizabeth A De Stasio. New Scientist. An excellent resource for the classroom teacher is Genetic Fingerprinting.. Vol 35. Pauline Lowrie and Susan Wells. 3.. 6. 2007. RFLP means restriction fragment length polymorphisms. Peter Neufeld and Nevelle Coleman. Scientific American. Individuals possess variable restriction recognition sites so that two pieces of DNA from separate sources may have different fragment lengths when their DNA is cut by the same enzyme. 4. 2. No 3.Appendix F References 1. Lauren Walsh. Pamela Zurer.
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